Liquid-liquid phase separation (LLPS) is a significant biophysical phenomenon that plays a crucial role in numerous pathophysiological processes associated with tumors. This study aims to establish a prognostic and immunotherapeutic response prediction signature for clear cell renal cell carcinoma (ccRCC) based on LLPS-related genes. Firstly, 66 differentially expressed LLPS-related genes were obtained from the TCGA, and then the LLPS prognostic signature constructed from five genes, including HOXA13, TEAD4, KMT5C, TRNP1, and SGO1, was identified by univariate Cox and least absolute shrinkage and selection operator algorithm. Meanwhile, the results of Kaplan-Meier analysis, ROC curves, principal component analysis, and nomogram showed high reliability of the constructed LLPS signature. Additionally, the relationship between the signature and immune infiltration, immunotherapy, and sensitivity to targeted therapeutic agents was discussed in this study. Single-cell sequencing data were used to analyze the expression levels of the signature genes in different cells in the ccRCC microenvironment. Finally, the expression of the signature genes was verified in ccRCC cells, and the effect of SGO1 on the malignant biological behavior of ccRCC cells was also verified by in vitro experiments. In conclusion, this LLPS prognostic signature may be a potential clinical prognostic and immunotherapeutic response-related indicator for ccRCC patients.

FET proteins are large multifunctional proteins that have several key roles in biology. The FET family of proteins, including FUS, EWSR1, and TAF15, play critical roles in transcription regulation, RNA processing, and DNA damage repair. These multifunctional RNA- and DNA-binding proteins are ubiquitously expressed and conserved across vertebrate species. They contain low-complexity (LC) domains that allow them to assemble and phase separate but also makes the proteins prone to aggregation. Aberrations in FET proteins, such as point mutations, aggregation, or translocations leading to fusion proteins, have been implicated in several pathologies, including frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), and Ewing sarcoma. In vitro study of FET proteins is hampered by their propensity to aggregate, their disordered structure, and their susceptibility to proteolysis, making high-yield production difficult. Here, we present optimized methods for the purification of full-length FUS, EWSR1, and their fusion proteins. These protocols enable researchers to overcome issues related to aggregation and solubility, facilitating biochemical and biophysical studies of these critical yet complex proteins. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Purification of EWSR1 and FUS proteins Alternate Protocol: Purification for fusion proteins.

Growing evidence indicates that abnormal liquid-liquid phase separation (LLPS) can disrupt biomolecular condensates, contributing to cancer development and progression. However, the influence of LLPS on the prognosis of head and neck squamous cell carcinoma (HNSCC) patients and its effects on the tumor immune microenvironment (TIME) are not yet fully understood. Therefore, we aimed to categorize patients with HNSCC based on LLPS-related genes and explored their multidimensional heterogeneity.

We integrated the transcriptomic data of 3,541 LLPS-related genes to assess the LLPS patterns in 501 patients with HNSCC within The Cancer Genome Atlas cohort. Subsequently, we explored the differences among the three LLPS subtypes using multi-omics analysis. We also developed an LLPS-related prognostic risk signature (LPRS) to facilitate personalized and integrative assessments and then screened and validated potential therapeutic small molecule compounds targeting HNSCC via experimental analyses.

By analyzing the expression profiles of 85 scaffolds, 355 regulators, and 3,101 clients of LLPS in HNSCC, we identified three distinct LLPS subtypes: LS1, LS2, and LS3. We confirmed notable differences among these subtypes in terms of prognosis, functional enrichment, genomic alterations, TIME patterns, and responses to immunotherapy. Additionally, we developed the LPRS, a prognostic signature for personalized integrative assessments, which demonstrated strong predictive capability for HNSCC prognosis across multiple cohorts. The LPRS also showed significant correlations with the clinicopathological features and TIME patterns in HNSCC patients. Furthermore, the LPRS effectively predicted responses to immune checkpoint inhibitor therapy and facilitated the screening of potential small-molecule compounds for treating HNSCC patients.

This study presents a new classification system for HNSCC patients grounded in LLPS. The LPRS developed in this research offers improved personalized prognosis and could optimize immunotherapy strategies for HNSCC.

Recurrent cancer-causing fusions of NUP98 produce higher-order assemblies known as condensates. How NUP98 oncofusion-driven condensates activate oncogenes remains poorly understood. Here, we investigate NUP98-PHF23, a leukemogenic chimera of the disordered phenylalanine-and-glycine (FG)-repeat-rich region of NUP98 and the H3K4me3/2-binding plant homeodomain (PHD) finger domain of PHF23. Our integrated analyses using mutagenesis, proteomics, genomics, and condensate reconstitution demonstrate that the PHD domain targets condensate to the H3K4me3/2-demarcated developmental genes, while FG repeats determine the condensate composition and gene activation. FG repeats are necessary to form condensates that partition a specific set of transcriptional regulators, notably the KMT2/MLL H3K4 methyltransferases, histone acetyltransferases, and BRD4. FG repeats are sufficient to partition transcriptional regulators and activate a reporter when tethered to a genomic locus. NUP98-PHF23 assembles the chromatin-bound condensates that partition multiple positive regulators, initiating a feedforward loop of reading-and-writing the active histone modifications. This network of interactions enforces an open chromatin landscape at proto-oncogenes, thereby driving cancerous transcriptional programs.

Cells are compartmentalized into different organelles to ensure precise spatial temporal control and efficient operation of cellular processes. Membraneless organelles, also known as biomolecular condensates, are emerging as previously underappreciated ways of organizing cellular functions. Condensates allow local concentration of protein, RNA, or DNA molecules with shared functions, thus facilitating spatiotemporal control of biochemical reactions spanning a range of cellular processes. Studies discussed herein have shown that aberrant formation of condensates is associated with various diseases such as cancers. Here, we summarize how condensates mechanistically contribute to malignancy-related cellular processes, including genomic instability, epigenetic rewiring, oncogenic transcriptional activation, and signaling. An improved understanding of condensate formation and dissolution will enable development of new cancer therapies. Finally, we address the remaining challenges in the field and suggest future efforts to better integrate condensates into cancer research.

Cells orchestrate their processes through complex interactions, precisely organizing biomolecules in space and time. Recent discoveries have highlighted the crucial role of biomolecular condensates-membrane-less assemblies formed through the condensation of proteins, nucleic acids, and other molecules-in driving efficient and dynamic cellular processes. These condensates are integral to various physiological functions, such as gene expression and intracellular signal transduction, enabling rapid and finely tuned cellular responses. Their ability to regulate cellular signaling pathways is particularly significant, as it requires a careful balance between flexibility and precision. Disruption of this balance can lead to pathological conditions, including neurodegenerative diseases, cancer, and viral infections. Consequently, biomolecular condensates have emerged as promising therapeutic targets, with the potential to offer novel approaches to disease treatment. In this review, we present the recent insights into the regulatory mechanisms by which biomolecular condensates influence intracellular signaling pathways, their roles in health and disease, and potential strategies for modulating condensate dynamics as a therapeutic approach. Understanding these emerging principles may provide valuable directions for developing effective treatments targeting the aberrant behavior of biomolecular condensates in various diseases.

Liquid-liquid phase separation (LLPS) drives the formation of membraneless intracellular compartments within both cytoplasm and nucleus. These compartments can form distinct physicochemical environments, and in particular display different concentrations of proteins, RNA, and macromolecules compared to the surrounding cytosol. Recent studies have highlighted the significant role of aberrant LLPS in cancer development and progression, impacting many core processes such as oncogenic signalling pathways, transcriptional dysregulation, and genome instability. In hepatocellular carcinoma (HCC), aberrant formation of biomolecular condensates has been observed in a number of preclinical models, highlighting their significance as an emerging factor in understanding cancer biology and its molecular underpinnings. In this review, we summarize emerging evidence and recent advances in understanding the role of LLPS in HCC, with a particular focus on the regulation and dysregulation of cytoplasmic and nuclear condensates in cancer cells. We finally discuss how an emerging understanding of phase separation processes in HCC opens up new potential treatment avenues.

Senescent cells are in a stable state of cell cycle arrest, leading to a natural barrier to tumorigenesis. Senescent cells secrete a pool of molecules, including cytokines, chemokines, proteases, and growth factors, termed the senescence-associated secretory phenotype (SASP), paradoxically contributing to pro-tumorigenic processes. However, the mechanism for regulating senescence and SASP in tumor cells remains unclear. Here, SPiDER senescence probe-based CRISPR/Cas9 library screening has identified ETS homologous factor (EHF) could effectively induce cellular senescence but without SASP, which could further significantly inhibit PDAC progression. Mechanically, tumoral EHF could form liquid-like condensates and further transcriptionally repress the expression of telomerase reverse transcriptase (TERT) and associated inflammatory factors, such as IL-6, CXCL12, etc. The reduction of TERT led to the telomere shortening and dysfunction of cancer cells, which further drove cellular senescence in PDAC. Moreover, EHF-mediated repression of inflammatory factors effectively declined the infiltration of immunosuppressive cells including MDSCs, Tregs, neutrophils, and promoted the accumulation of CD8+T cells and NK cells, which enhanced tumor immune surveillance. Furthermore, high throughput drug screening identified that Bilobetin could effectively promote the phase separation of EHF, which could further induce tumoral senescence but without SASP. In vivo, preclinical translational research uncovered that Bilobetin could ameliorate immunosuppressive tumor microenvironment (TME) and sensitize PDAC to anti-PD-1 therapy. Overall, our study revealed EHF as a potential candidate to overcome the paradoxical function of cellular senescence and elucidated the effects of its phase separation state on gene regulation, which provided new insights and strategies for PDAC treatment.

Drug resistance remains a challenge for targeted therapy of cancers driven by EML4-ALK and related fusion oncogenes. EML4-ALK forms cytoplasmic protein condensates, which result from networks of interactions between oncogene and adapter protein multimers. While these assemblies are associated with oncogenic signaling, their role in drug response is unclear. Here, we use optogenetics and live-cell imaging to find that EML4-ALK assemblies suppress transmembrane receptor tyrosine kinase (RTK) signaling by sequestering RTK adapter proteins including GRB2 and SOS1. Furthermore, ALK inhibition, while suppressing oncogenic signaling, simultaneously releases the sequestered adapters and thereby resensitizes RTK signaling. Resensitized RTKs promote rapid and pulsatile ERK reactivation that originates from paracrine ligands shed by dying cells. Reactivated ERK signaling promotes cell survival, which can be counteracted by combination therapies that block paracrine signaling. Our results identify a regulatory role for RTK fusion assemblies and uncover a mechanism of tolerance to targeted therapies.

Biomolecular condensates are increasingly recognized as important drivers of cellular function; their dysregulation leads to pathology and disease. We discuss three questions in terms of the impending utility of data-driven techniques to predict condensate-driven biological outcomes, i.e., the impact of cellular state changes on condensates, the effect of condensates on biochemical processes within, and condensate properties that result in cellular dysregulation and disease.

The nanoscale organization of enzymes associated with the dynamics of second messengers is critical for ensuring compartmentation and localization of signaling molecules in cells. Specifically, the spatiotemporal orchestration of cAMP and Ca2+ oscillations is critical for many cellular functions. Previous experimental studies have shown that the formation of nanodomains of A-kinase anchoring protein 79/150 (AKAP150) and adenylyl cyclase 8 (AC8) on the surface of pancreatic MIN6 β cells modulates the phase of Ca2+-cAMP oscillations from out-of-phase to in-phase. In this work, we develop computational models of the Ca2+/cAMP pathway and AKAP/AC nanodomain formation that give rise to the two important predictions: instead of an arbitrary phase difference, the out-of-phase Ca2+/cAMP oscillation reaches Ca2+ trough and cAMP peak simultaneously, which is defined as inversely out-of-phase; the in-phase and inversely out-of-phase oscillations associated with Ca2+-cAMP dynamics on and away from the nanodomains can be explained by an incoherent feedforward loop. Factors such as cellular surface-to-volume ratio, compartment size, and distance between nanodomains do not affect the existence of in-phase or inversely out-of-phase Ca2+/cAMP oscillation, but cellular surface-to-volume ratio and compartment size can affect the time delay for the inversely out-of-phase Ca2+/cAMP oscillation while the distance between two nanodomains does not. Finally, we predict that both the Turing pattern-generated nanodomains and experimentally measured nanodomains demonstrate the existence of in-phase and inversely out-of-phase Ca2+/cAMP oscillation when the AC8 is at a low level, consistent with the behavior of an incoherent feedforward loop. These findings unveil the key circuit motif that governs cAMP and Ca2+ oscillations and advance our understanding of how nanodomains can lead to spatial compartmentation of second messengers.

Biomolecular condensates are dynamic membraneless organelles that compartmentalize proteins and RNA molecules to regulate key cellular processes. Diverse RNA species exert their effects on the cell by their roles in condensate formation and function. RNA abnormalities such as overexpression, modification, and mislocalization can lead to pathological condensate behaviors that drive various diseases, including cancer, neurological disorders, and infections. Here, we review RNA's role in condensate biology, describe the mechanisms of RNA-induced condensate dysregulation, note the implications for disease pathogenesis, and discuss novel therapeutic strategies. Emerging approaches to targeting RNA within condensates, including small molecules and RNA-based therapies that leverage the unique properties of condensates, may revolutionize treatment for complex diseases.

Cellular compartmentalization, achieved through membrane-based compartments, is a fundamental aspect of cell biology that contributes to the evolutionary success of cells. While organelles have traditionally been the focus of research, membrane-less organelles (MLOs) are emerging as critical players, exhibiting distinct morphological features and unique molecular compositions. Recent research highlights the pivotal role of long noncoding RNAs (lncRNAs) in MLOs and their involvement in various cellular processes across different organisms. In the context of cancer, dysregulation of MLO formation, influenced by altered lncRNA expression, impacts chromatin organization, oncogenic transcription, signaling pathways, and telomere lengthening. This review synthesizes the current understanding of lncRNA composition within MLOs, delineating their functions and exploring how their dysregulation contributes to human cancers. Environmental challenges in tumorigenesis, such as nutrient deprivation and hypoxia, induce stress granules, promoting cancer cell survival and progression. Advancements in biochemical techniques, particularly single RNA imaging methods, offer valuable tools for studying RNA functions within live cells. However, detecting low-abundance lncRNAs remains challenging due to their limited expression levels. The correlation between lncRNA expression and pathological conditions, particularly cancer, should be explored, emphasizing the importance of single-cell studies for precise biomarker identification and the development of personalized therapeutic strategies. This article is categorized under: RNA Export and Localization > RNA Localization RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

YAP/TEAD signaling is essential for organismal development, cell proliferation, and cancer progression. As a transcriptional coactivator, how YAP activates its downstream target genes is incompletely understood. YAP forms biomolecular condensates in response to hyperosmotic stress, concentrating transcription-related factors to activate downstream target genes. However, whether YAP forms condensates under other signals, how YAP condensates organize and function, and how YAP condensates activate transcription in general are unknown. Here, we report that endogenous YAP forms sub-micron scale condensates in response to Hippo pathway regulation and actin cytoskeletal tension. YAP condensates are stabilized by the transcription factor TEAD1, and recruit BRD4, a coactivator that is enriched at active enhancers. Using single-particle tracking, we found that YAP condensates slowed YAP diffusion within condensate boundaries, a possible mechanism for promoting YAP target search. These results reveal that YAP condensate formation is a highly regulated process that is critical for YAP/TEAD target gene expression.

Gastric cancer is a frequent and lethal solid tumor that has a poor prognosis and treatment result. Reprogramming of nucleotide metabolism is a characteristic of cancer development and progression.

We used a variety of machine learning techniques to create a novel nucleotide metabolism-related index (NMRI) using gastric cancer sample data obtained from the TCGA and GEO databases. This index is based on genes associated to nucleotide metabolism. Gastric cancer patients were categorized into high and low NMRI groups based on NMRI results. The clinical features, tumor immune microenvironment, response to chemotherapy, and response to immunotherapy were then thoroughly examined. In vitro experiments were then used to confirm the biological role of SERPINE1 in gastric cancer.

The four nucleotide metabolism-related genes that make up NMRI (GAMT, ORC1, CNGB3, and SERPINE1) were verified in an external dataset and are a valid predictor of prognosis for patients with gastric cancer. The high NMRI group was more responsive to immunotherapy and had greater levels of immune cell infiltration than the low NMRI group. The proliferation and migration of stomach cancer was shown to be decreased by SERPINE1 knockdown in vitro.

This study's NMRI can reliably predict a patient's prognosis for stomach cancer and pinpoint the patient group that will benefit from immunotherapy, offering important new information on the clinical treatment of stomach cancer.

RNA-binding proteins (RBPs) are pivotal in acute myeloid leukaemia (AML), a lethal disease. Although specific phase separation-competent RBPs are recognized in AML, the effect of their condensate formation on AML leukaemogenesis, and the therapeutic potential of inhibition of phase separation are underexplored. In our in vivo CRISPR RBP screen, fibrillarin (FBL) emerges as a crucial nucleolar protein that regulates AML cell survival, primarily through its phase separation domains rather than methyltransferase or acetylation domains. These phase separation domains, with specific features, coordinately drive nucleoli formation and early processing of pre-rRNA (including efflux, cleavage and methylation), eventually enhancing the translation of oncogenes such as MYC. Targeting the phase separation capability of FBL with CGX-635 leads to elimination of AML cells, suggesting an additional mechanism of action for CGX-635 that complements its established therapeutic effects. We highlight the potential of PS modulation of critical proteins as a possible therapeutic strategy for AML.

Receptor tyrosine kinase (RTK)-mediated signal transduction is fundamental to cell function and drives important cellular outcomes which, when dysregulated, can lead to malignant tumour growth and metastasis. The initiation of signals from plasma membrane-bound RTKs is subjected to multiple regulatory mechanisms that control downstream effector protein recruitment and function. The high propensity of RTKs to condense via liquid-liquid phase separation (LLPS) into membraneless organelles with downstream effector proteins provides a further fundamental mechanism for signal regulation. Herein we highlight how this phenomenon contributes to cancer signalling and consider the potential impact of LLPS on outcomes for cancer patients.

Dysregulated liquid-liquid phase separation (LLPS) instigates tumorigenesis through biomolecular condensate dysfunction. However, the association between LLPS-associated genes and glioma remains underexplored.

Differentially expressed genes (DEGs) of glioma were obtained from the GSE50161 dataset, including 34 glioma and 13 normal samples. We analyzed differentially expressed LLPS-related genes in glioma from public databases. These genes informed refined molecular subtyping on the TCGA-glioma dataset. CIBERSORT assessed immune cell infiltration across three subclusters. A prognostic model was devised using univariate and lasso Cox regressions on intersecting genes. Prognostic gene expression was validated in glioma cells via RT-qPCR.

A total of 673 differentially expressed LLPS-associated genes were identified in glioma. Three distinct molecular subtypes (C1, C2, and C3) of glioma were obtained with a marked variance in the expression of immune checkpoint genes PD1 and PDL1. Differences in immune cell infiltration were observed across subtypes. In addition, a tri-gene prognostic signature (TAGLN2, NTNG2, and IGF2BP2) was derived with significant survival differences between high and low-risk groups. The prognostic model displayed impressive AUC values for 1, 3, and 5-year survival in both training and validation sets. Further analysis highlighted a notable correlation between the three prognostic genes and immune cells in glioma samples. Furthermore, we found the upregulation of TAGLN2 and IGF2BP2 and the downregulation of NTNG2 in glioma tumors and cells.

This study innovatively uncovers the significant role of LLPS-related genes in glioma tumor grading and prognosis. The constructed tri-gene prognostic model holds promise for enhancing personalized prognosis assessments and optimizing immunotherapy strategies for glioma patients.

Beyond lipid membrane compartments, cells including cancer cells utilize various membraneless compartments, often termed biomolecular condensates, to regulate or organize key cellular processes underlying physiologic or pathologic phenotypes. In this commentary, the emergence of biomolecular condensation in cancer biology is highlighted, with a focus on key unanswered questions and with implications for improving the understanding of cancer pathogenesis and developing innovative cancer management strategies.

Liquid-liquid phase separation (LLPS) refers to the phenomenon, where a homogeneous solution spontaneously undergoes a transition into two or more immiscible phases. Through transient weak multivalent macromolecular interactions, a homogeneous solution can spontaneously separate into two phases: one rich in biomolecules and the other poor in biomolecules. Phase separation is believed to serve as the physicochemical foundation for the formation of membrane-less organelles (MLOs) and bio-molecular condensates within cells. Moreover, numerous biological processes depend on LLPS, such as transcription, immunological response, chromatin architecture, DNA damage response, stress granule formation, viral infection, etc. Abnormalities in phase separation can lead to diseases, such as cancer, neurodegeneration, and metabolic disorders. LLPS is regulated by various factors, such as concentration of molecules undergoing LLPS, salt concentration, pH, temperature, post-translational modifications, and molecular chaperones. Recent research on LLPS of biomolecules has progressed rapidly and led to the development of databases containing information pertaining to various aspects of the biomolecule separation analysis. However, more comprehensive research is still required to fully comprehend the specific molecular mechanisms and biological effects of LLPS.

The pathological aggregation and misfolding of tau and amyloid-β play a key role in Alzheimer's disease (AD). However, the underlying pathological mechanisms remain unclear. Emerging evidences indicate that liquid-liquid phase separation (LLPS) has great impacts on regulating human health and diseases, especially neurodegenerative diseases. A series of studies have revealed the significance of LLPS in AD. In this review, we summarize the latest progress of LLPS in AD, focusing on the impact of metal ions, small-molecule inhibitors, and proteinaceous partners on tau LLPS and aggregation, as well as toxic oligomerization, the role of LLPS on amyloid-β (Aβ) aggregation, and the cross-interactions between amyloidogenic proteins in AD. Eventually, the fundamental methods and techniques used in LLPS study are introduced. We expect to present readers a deeper understanding of the relationship between LLPS and AD.

Liquid-liquid phase separation (LLPS) in biology describes a process by which proteins form membraneless condensates within a cellular compartment when conditions are met, including the concentration and posttranslational modifications of the protein components, the condition of the aqueous solution (pH, ionic strength, pressure, and temperature), and the existence of assisting factors (such as RNAs or other proteins). In these supramolecular liquid droplet-like inclusion bodies, molecules are held together through weak intermolecular and/or intramolecular interactions. With the aid of LLPS, cells can assemble functional sub-units within a given cellular compartment by enriching or excluding specific factors, modulating cellular function, and rapidly responding to environmental or physiological cues. Hence, LLPS is emerging as an important means to regulate biology and physiology. Yet, excessive inclusion body formation by, for instance, higher-than-normal concentrations or mutant forms of the protein components could result in the conversion from dynamic liquid condensates into more rigid gel- or solid-like aggregates, leading to the disruption of the organelle's function followed by the development of human disorders like neurodegenerative diseases. In summary, well-controlled formation and de-formation of LLPS is critical for normal biology and physiology from single cells to individual organisms, whereas abnormal LLPS is involved in the pathophysiology of human diseases. In turn, targeting these aggregates or their formation represents a promising approach in treating diseases driven by abnormal LLPS including those neurodegenerative diseases that lack effective therapies.

VEGFR-2 tyrosine kinase inhibitors are receiving a lot of attention as prospective anticancer medications in the current drug discovery process.

This work aims to explore the PubChem library for novel VEGFR-2 kinase inhibitors. 1H-Indazole-containing drug AXITINIB, or AG-013736 (FDA approved), is chosen as a rational molecule for drug design. This scaffold proved its efficiency in treating cancer and other diseases as well.

The present study used the virtual screening of the database, protein preparation, grid creation, and molecular docking analyses.

The protein was validated on different parameters like the Ramachandran plot, the ERRAT score, and the ProSA score. The Ramachandran plot revealed that 92.1% of the amino acid residues were located in the most favorable region; this was complemented by an ERRAT score (overall quality factor) of 96.24 percent and a ProSA (Z score) of -9.24 percent. The Lipinski rule of five was used as an additional filter for screening molecules. The docking results showed values of binding affinity between -14.08 and -12.34 kcal/mol. The molecule C1 showed the highest docking value of -14.08 Kcal/mol with the maximum number of strong H-bonds by -NH of pyridine to amino acid Cys104 (4.22Å), -NH of indazole to Glu108 (4.72), and Glu70 to bridge H of -NH. These interactions are similar to Axitinib docking interactions like Glu70, Cys104, and Glu102. The docking studies revealed that pi-alkyl bonds are formed with unsubstituted pyridine, whereas important H-bonds are observed with different substitutions around -NH. Based on potential findings, we designed new molecules, and molecular docking studies were performed on the same protein along with ADMET studies. The designed molecules (M1-M4) also showed comparable docking results similar to Axitinib, along with a synthetic accessibility score of less than 4.5.

The docking method employed in this work opens up new possibilities for the design and synthesis of novel compounds that can act as VEGFR-2 tyrosine kinase inhibitors and treat cancer.

Liquid-liquid phase separation (LLPS) is a novel principle for interpreting precise spatiotemporal coordination in living cells through biomolecular condensate (BMC) formation via dynamic aggregation. LLPS changes individual molecules into membrane-free, droplet-like BMCs with specific functions, which coordinate various cellular activities. The formation and regulation of LLPS are closely associated with oncogenesis, tumor progressions and metastasis, the specific roles and mechanisms of LLPS in tumors still need to be further investigated at present. In this review, we comprehensively summarize the conditions of LLPS and identify mechanisms involved in abnormal LLPS in cancer processes, including tumor growth, metastasis, and angiogenesis from the perspective of cancer hallmarks. We have also reviewed the clinical applications of LLPS in oncologic areas. This systematic summary of dysregulated LLPS from the different dimensions of cancer hallmarks will build a bridge for determining its specific functions to further guide basic research, finding strategies to intervene in LLPS, and developing relevant therapeutic approaches.

Transcription is the fundamental process of gene expression, which in eukaryotes occurs within the complex physicochemical environment of the nucleus. Decades of research have provided extreme detail in the molecular and functional mechanisms of transcription, but the spatial and genomic organization of transcription remains mysterious. Recent discoveries show that transcriptional components can undergo phase separation and create distinct compartments inside the nucleus, providing new models through which to view the transcription process in eukaryotes. In this review, we focus on transcriptional condensates and their phase separation-like behaviors. We suggest differentiation between physical descriptions of phase separation and the complex and dynamic biomolecular assemblies required for productive gene expression, and we discuss how transcriptional condensates are central to organizing the three-dimensional genome across spatial and temporal scales. Finally, we map approaches for therapeutic manipulation of transcriptional condensates and ask what technical advances are needed to understand transcriptional condensates more completely.

Liquid-liquid phase separation (LLPS) is characterized as an ubiquitous framework for diverse biological processes including carcinogenesis and cancer progression. While targeting cancer from perspective of LLPS offers an opportunity to drug the conventionally undruggables with cancer-driving potential, the therapeutic value of cancer associated LLPS (CAL) proteins remains elusive. Here, we report the genomic landscape, prognostic relevance, immune-infiltration association, down-stream pathway alteration and small molecular responsiveness of CAL protein-coding gene signatures based on protein-coding associated mutations and transcriptional abundance in pan-cancer. Correlations of CAL protein-coding associated mutations and transcriptional abundances to overall survival and progression-free survival were observed in an array of cancers and further characterized by differential survival outcomes between patients with intrinsic disordered region (IDR) enriched and non-IDR enriched mutations in endometrial cancer. Altered signaling pathways and universal pattern of immune infiltrates on account of CAL protein-coding associated gene-set mutations involved key components of oncogenesis in various cancer types and well established therapeutic targets including MAPK signaling pathway and implied an inflamed tumor immunity that might be highly responsive to immunotherapy. LLPS inhibitor enhanced cytotoxicity of cisplatin/paclitaxel in selective cancer cell lines. These findings provide preliminary evidences for rational chemo-, targeted- and immuno-therapeutic innovation with LLPS regulating synergy.

Non-coding RNAs have shown key roles in cancer and among them, short RNA molecules are known as microRNAs (miRNAs). These molecules have length less than 25 nucleotides and suppress translation and expression. The functional miRNAs are produced in cytoplasm. Lung cancer is a devastating disease that its mortality and morbidity have undergone an increase in recent years. Aggressive behavior leads to undesirable prognosis and tumors demonstrate abnormal proliferation and invasion. In the present review, miRNA functions in lung cancer is described. miRNAs reduce/increase proliferation and metastasis. They modulate cell death and proliferation. Overexpression of oncogenic miRNAs facilitates drug resistance and radio-resistance in lung cancer. Tumor microenvironment components including macrophages and cancer-associated fibroblasts demonstrate interactions with miRNAs in lung cancer. Other factors such as HIF-1α, lncRNAs and circRNAs modulate miRNA expression. miRNAs have also value in the diagnosis of lung cancer. Understanding such interactions can pave the way for developing novel therapeutics in near future for lung cancer patients.

Subcellular mRNA localization is critical to a multitude of biological processes such as development of cellular polarity, embryogenesis, tissue differentiation, protein complex formation, cell migration, and rapid responses to environmental stimuli and synaptic depolarization. Our understanding of the mechanisms of mRNA localization must now be revised to include formation and trafficking of biomolecular condensates, as several biomolecular condensates that transport and localize mRNA have recently been discovered. Disruptions in mRNA localization can have catastrophic effects on developmental processes and biomolecular condensate biology and have been shown to contribute to diverse diseases. A fundamental understanding of mRNA localization is essential to understanding how aberrations in this biology contribute the etiology of numerous cancers though support of cancer cell migration and biomolecular condensate dysregulation, as well as many neurodegenerative diseases, through misregulation of mRNA localization and biomolecular condensate biology. This article is categorized under: RNA Export and Localization > RNA Localization RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development.

RAP80 has been characterized as a component of the BRCA1-A complex and is responsible for the recruitment of BRCA1 to DNA double-strand breaks (DSBs). However, we and others found that the recruitment of RAP80 and BRCA1 were not absolutely temporally synchronized, indicating that other mechanisms, apart from physical interaction, might be implicated. Recently, liquid-liquid phase separation (LLPS) has been characterized as a novel mechanism for the organization of key signaling molecules to drive their particular cellular functions. Here, we characterized that RAP80 LLPS at DSB was required for RAP80-mediated BRCA1 recruitment. Both cellular and in vitro experiments showed that RAP80 phase separated at DSB, which was ascribed to a highly disordered region (IDR) at its N-terminal. Meanwhile, the Lys63-linked poly-ubiquitin chains that quickly formed after DSBs occur, strongly enhanced RAP80 phase separation and were responsible for the induction of RAP80 condensation at the DSB site. Most importantly, abolishing the condensation of RAP80 significantly suppressed the formation of BRCA1 foci, encovering a pivotal role of RAP80 condensates in BRCA1 recruitment and radiosensitivity. Together, our study disclosed a new mechanism underlying RAP80-mediated BRCA1 recruitment, which provided new insight into the role of phase separation in DSB repair.

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4+ T cell nuclear lysates led to the formation of larger BMCs compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggest that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.

In recent years, there has been growing evidence indicating a relationship between liquid-liquid phase separation (LLPS) and cancer development. However, to date, the clinical significance of LLPS in skin cutaneous melanoma (SKCM, hereafter referred to as melanoma) remains to be elucidated. In the current study, the impact of LLPS-related genes on melanoma prognosis has been explored.

LLPS-related genes were retrieved from the DrLLPS database. The prognostic feature for LLPS in melanoma was developed in The Cancer Genome Atlas (TCGA) dataset and verified in the GSE65904 cohort. Based on risk scores, melanoma patients were categorized into high- and low-risk groups. Thereafter, the differences in clinicopathological correlation, functional enrichment, immune landscape, tumor mutational burden, and impact of immunotherapy between the two groups were investigated. Finally, the role of key gene TROAP in melanoma was validated by in vitro and in vivo experiments.

The LLPS-related gene signature was developed based on MLKL, PARVA, PKP1, PSME1, RNF114, and TROAP. The risk score was a crucial independent prognostic factor for melanoma and patients with high-risk scores were related to a worse prognosis. Approximately, all immune-relevant characteristics, such as immune cell infiltration and immune scores, were extremely evident in patients with low-risk scores. The findings from the in vitro and in vivo experiments indicated that the viability, proliferation, and invasion ability of melanoma cells were drastically decreased after the knockdown of TROAP.

Our gene signature can independently predict the survival of melanoma patients. It provides a basis for the exploration of the relationship between LLPS and melanoma and can offer a fresh perspective on the clinical diagnosis and treatment of the disease.

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that pan-retroviral nucleocapsid (NC) and the HIV-1 pr55 ^Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yield self-assembling BMCs having HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4 ⁺ T cell nuclear lysates led to the formation of larger BMCs as compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggests that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.

The unprecedented non-reproducibility of the results published in the field of cancer research has recently come under the spotlight. In this short review, we try to highlight some general principles in the organization and evolution of cancerous tumors, which objectively lead to their enormous variability and, consequently, the irreproducibility of the results of their investigation. This heterogeneity is also extremely unfavorable for the effective use of molecularly targeted medicine. Against the seemingly comprehensive background of this heterogeneity, we single out two supramolecular characteristics common to all tumors: the clustered nature of tumor interactions with their microenvironment and the formation of biomolecular condensates with tumor-specific distinctive features. We suggest that these features can form the basis of strategies for tumor-specific supramolecular targeted therapies.

Transcriptional deregulation, a cancer cell hallmark, is driven by epigenetic abnormalities in the majority of brain tumors, including adult glioblastoma and pediatric brain tumors. Epigenetic abnormalities can activate epigenetic regulatory elements to regulate the expression of oncogenes. Superenhancers (SEs), identified as novel epigenetic regulatory elements, are clusters of enhancers with cell-type specificity that can drive the aberrant transcription of oncogenes and promote tumor initiation and progression. As gene regulators, SEs are involved in tumorigenesis in a variety of tumors, including brain tumors. SEs are susceptible to inhibition by their key components, such as bromodomain protein 4 and cyclin-dependent kinase 7, providing new opportunities for antitumor therapy. In this review, we summarized the characteristics and identification, unique organizational structures, and activation mechanisms of SEs in tumors, as well as the clinical applications related to SEs in tumor therapy and prognostication. Based on a review of the literature, we discussed the relationship between SEs and different brain tumors and potential therapeutic targets, focusing on glioblastoma.

Cancer growth, annexation, and metastatic spread are all aided by the formation of new blood vessels (angiogenesis). The commencement of the VEGF pathway leads to signal transduction that enhances endothelial cell survival, relocation, and divergence from pre-existing vasculature. The ability of solid malignancies to bloom and spread depends critically on their ability to establish their independent blood circulation (tumor angiogenesis). VEGFR is a major receptor tyrosine kinase that regulates angiogenesis, cell growth, and metastasis, diminishing apoptosis, cytoskeletal function, and other biological processes VEGFR has proven to be a remarkable focus for a variety of anticancer medicines in clinical studies. This Review explores the development of anti-VEGF-based antiangiogenic therapies having different scaffolds. This review had focused on SAR and docking studies of previously reported molecules.

Mounting evidence has demonstrated that an imbalance in liquid-liquid phase separation (LLPS) can induce alteration in the spatiotemporal coordination of biomolecular condensates, which plays a role in carcinogenesis and cachexia. However, the role of LLPS in the occurrence and progression of bladder cancer (BLCA) remains to be elucidated. Identifying the role of LLPS in carcinogenesis may aid in cancer therapeutics.

A total of 1,351 BLCA samples from six cohorts were retrieved from publicly available databases like The Cancer Genome Atlas, Gene Expression Omnibus, and ArrayExpress. The samples were divided into three distinct clusters, and their multi-dimensional heterogeneities were explored. The LLPS patterns of all patients were determined based on the LLPS-related risk score (LLPSRS), and its multifaceted landscape was depicted and experimentally validated at the multi-omics level. Finally, a cytotoxicity-related and LLPSRS-based classifier was established to predict the patient's response to immune checkpoint blockade (ICB) treatment.

Three LLPS-related subtypes were identified and validated. The differences in prognosis, tumor microenvironment (TME) features, cancer hallmarks, and certain signatures of the three LLPS-related subtypes were validated. LLPSRS was calculated, which could be used as a prognostic biomarker. A close correlation was observed between clinicopathological features, genomic variations, biological mechanisms, immune infiltration in TME, chemosensitivity, and LLPSRS. Furthermore, our classifier could effectively predict immunotherapy response in patients with BLCA.

Our study identified a novel categorization of BLCA patients based on LLPS. The LLPSRS could predict the prognosis of patients and aid in designing personalized medicine. Further, our binary classifier could effectively predict patients' sensitivity to immunotherapy.

Growing evidence suggests prevalence of transcriptional condensates on chromatin, yet their mechanisms of formation and functional significance remain largely unclear. In human cancer, a series of mutations in the histone acetylation reader ENL create gain-of-function mutants with increased transcriptional activation ability. Here, we show that these mutations, clustered in ENL's structured acetyl-reading YEATS domain, trigger aberrant condensates at native genomic targets through multivalent homotypic and heterotypic interactions. Mechanistically, mutation-induced structural changes in the YEATS domain, ENL's two disordered regions of opposing charges, and the incorporation of extrinsic elongation factors are all required for ENL condensate formation. Extensive mutagenesis establishes condensate formation as a driver of oncogenic gene activation. Furthermore, expression of ENL mutants beyond the endogenous level leads to non-functional condensates. Our findings provide new mechanistic and functional insights into cancer-associated condensates and support condensate dysregulation as an oncogenic mechanism.

Biomolecular condensates play numerous roles in cells by selectively concentrating client proteins while excluding others. These functions are likely to be sensitive to the spatial organization of the scaffold proteins forming the condensate. We use coarse-grained molecular simulations to show that model intrinsically-disordered proteins phase separate into a heterogeneous, structured fluid characterized by a well-defined length scale. The proteins are modelled as semi-flexible polymers with punctate, multifunctional binding sites in good solvent conditions. Their dense phase is highly solvated with a spatial structure that is more sensitive to the separation of the binding sites than their affinity. We introduce graph theoretic measures to quantify their heterogeneity, and find that it increases with increasing binding site number, and exhibits multi-timescale dynamics. The model proteins also swell on passing from the dilute solution to the dense phase. The simulations predict that the structure of the dense phase is modulated by the location and affinity of binding sites distant from the termini of the proteins, while sites near the termini more strongly affect its phase behaviour. The relations uncovered between the arrangement of weak interaction sites on disordered proteins and the material properties of their dense phase can be experimentally tested to give insight into the biophysical properties, pathological effects, and rational design of biomolecular condensates.

Multicellularity was a watershed development in evolution. However, it also meant that individual cells could escape regulatory mechanisms that restrict proliferation at a severe cost to the organism: cancer. From the standpoint of cellular organization, evolutionary complexity scales to organize different molecules within the intracellular milieu. The recent realization that many biomolecules can "phase-separate" into membraneless organelles, reorganizing cellular biochemistry in space and time, has led to an explosion of research activity in this area. In this review, we explore mechanistic connections between phase separation and cancer-associated processes and emerging examples of how these become deranged in malignancy.

One of the fundamental functions of phase separation is to rapidly and dynamically respond to environmental perturbations. Importantly, these changes often lead to alterations in cancer-relevant pathways and processes. This review covers recent advances in the field, including emerging principles and mechanisms of phase separation in cancer.

Membraneless biomolecular condensates (BMCs) contribute to the replication of a growing number of viruses but remain to be functionally characterized. Previously, we demonstrated that pan-retroviral nucleocapsid (NC) proteins phase separated into condensates regulating virus assembly. Here we discover that intrinsically disordered human immunodeficiency virus-type 1 (HIV-1) core proteins condense with the viral genomic RNA (vRNA) to assemble as BMCs attaining a geometry characteristic of viral reverse transcription complexes. We explore the predisposition, mechanisms, and pharmacologic sensitivity of HIV-1 core BMCs in living cells. HIV-1 vRNA-interacting NC condensates were found to be scaffolds onto which client capsid, reverse transcriptase, and integrase condensates assemble. HIV-1 core BMCs exhibit fundamental characteristics of BMCs and are drug-sensitive. Lastly, protease-mediated maturation of Gag and Gag-Pol precursor proteins yield abundant and visible BMCs in cells. This study redefines HIV-1 core components as fluid BMCs and advances our understanding of the nature of viral cores during ingress.

Mechanical forces collaborate across length scales to coordinate cell fate during development and the dynamic homeostasis of adult tissues. Similarly, steroid hormones interact with their nuclear and nonnuclear receptors to regulate diverse physiological processes necessary for the appropriate development and function of complex multicellular tissues. Aberrant steroid hormone action is associated with tumors originating in hormone-sensitive tissues and its disruption forms the basis of several therapeutic interventions. Prolonged perturbations to mechanical forces may further foster tumor initiation and the evolution of aggressive metastatic disease. Recent evidence suggests that steroid hormone and mechanical signaling intersect to direct cell fate during development and tumor progression. Potential mechanosensitive steroid hormone signaling pathways along with their molecular effectors will be discussed in this context.