Biomolecular phase separation in tumorigenesis: from aberrant condensates to therapeutic vulnerabilities

Hu, Lan; Huang, Zikun; Liu, Zhaoyong; Zhang, Ying

doi:10.1186/s12943-025-02428-1

Review
Open access
Published: 23 August 2025

Biomolecular phase separation in tumorigenesis: from aberrant condensates to therapeutic vulnerabilities

Molecular Cancer volume 24, Article number: 220 (2025) Cite this article

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Abstract

Biomolecular phase separation has emerged as a fundamental mechanism governing intracellular spatial organization and functional compartmentalization, and is increasingly recognized as a critical factor in tumor initiation and progression. Through multivalent molecular interactions, biomolecular phase separation contributes to the formation of condensates that mediate the assembly of membraneless organelles, coordination of signaling pathways, and transcriptional programs. Under physiological conditions, condensation contributes to the maintenance of gene expression homeostasis, stress adaptation, and metabolic balance. In cancer cells, however, biomolecular condensates (BMCs) often exhibit aberrant behavior, accompanied by alterations in their structure, components, and regulatory mechanisms. Such perturbations may disrupt cellular homeostasis and influence key biological processes including gene regulation, signal transduction, metabolic reprogramming, and immune responses, thereby modulating various cancer hallmarks. Although the mechanistic understanding of BMCs remains incomplete, their intrinsic plasticity and environmental sensitivity make them attractive therapeutic targets for cancer treatment. This review provides a comprehensive overview of the regulatory factors and functional mechanisms of BMCs in cancer biology, with a particular focus on their involvement in diverse cancer hallmarks. This review further summarizes emerging therapeutic strategies targeting condensation, aiming to inspire novel treatment opportunities.

Introduction

Biomolecular phase separation has been widely recognized as a fundamental mechanism governing cellular compartmentalization. Phase separation describes the spontaneous segregation of molecules into dense and dilute phases, driving the formation of membraneless biomolecular condensates (BMCs) such as nucleoli and signaling clusters [1,2,3]. Condensation is mediated by multivalent interactions among intrinsically disordered regions (IDRs), RNA scaffolds, and modular domains [4,5,6,7]. This mechanism transcends classical membrane-bound compartmentalization, enabling precise regulation of biochemical reaction rates, specificity, and coordination [8]. BMCs most commonly arise through liquid-liquid phase separation (LLPS), yet their material properties are not confined to the liquid state. Depending on sequence characteristics, concentration, and environmental conditions, condensates can also exhibit viscoelastic or solid-like states, including gel and glass phases. Importantly, such physical states can emerge independently of aging or maturation processes and may contribute to either functional compartmentalization or pathological aggregation [9]. Studies have shown that although phase-separated condensates exhibit high macroscopic viscosity, biomolecular rearrangements remain rapid at the molecular scale, enabling efficient biochemical reactions [7, 10]. In vivo, such dynamic and reversible behavior facilitates rapid and accurate cellular responses to environmental changes, including transcriptional bursting, signal transduction, and genome architecture maintenance [11,12,13,14]. However, accumulating evidence indicates that condensates function not only as dynamic regulatory hubs but also as nucleation platforms for irreversible structural transitions under specific conditions. For instance, phase separation facilitates the condensation of actin or tubulin monomers, promoting the nucleation of filamentous structures [15,16,17,18,19]. Similarly, keratin and collagen undergo phase separation to promote the assembly of intermediate filaments and extracellular fibers [20, 21]. In such situations, condensation supports the formation of permanent structures. These findings reveal that biomolecular phase separation participates in both reversible regulation and irreversible structural transitions, raising the question of whether reversibility is a universal feature of condensates. In fact, growing evidence suggests that the reversible nature of phase separation is not a default state, but rather an evolutionarily selected property observed in many BMCs [22,23,24]. This dynamic property can be disrupted by mutations, environmental stress, or dysregulated signaling, rendering condensates susceptible to pathological perturbations. Accumulating evidence implicates aberrant condensate formation in various diseases, particularly neurodegenerative disorders and cancer. In the nervous system, proteins such as FUS and TDP-43 can form condensates via biomolecular phase separation, these condensates may develop into toxic aggregates that contribute to neurodegeneration [25, 26]. In cancer, mutant p53 forms irreversible dense condensates via LLPS, losing tumor-suppressive function while acquiring oncogenic properties [27].This review systematically describes the molecular mechanisms and regulatory factors of biomolecular phase separation, highlights its functional roles in diverse cancer hallmarks, and discusses emerging therapeutic strategies targeting condensates as well as the challenges and future directions.

The history of biomolecular phase separation

The research history of biomolecular phase separation can be traced to the 1830 s, when the nucleolus, a membrane-less organelle, was first observed, although its phase separation nature was not recognized at the time (Fig. 1). The modern era of biomolecular phase separation research began in 2009 when Hyman et al. made the seminal observation of P granule condensation and dissolution in C. elegans embryos, providing the first direct evidence implicating biomolecular phase separation as a mechanism of cellular compartmentalization [28]. This groundbreaking work challenges the traditional view that cellular organization relies on membrane-bound compartments. In 2010, Hyman et al. observed similar phase separation phenomena in the nucleolus of Xenopus laevis oocytes, further supporting the idea that biomolecular phase separation may represent a universal organizational strategy in cells. In 2012, biomolecular phase separation research entered a new era. Rosen et al. through in vitro reconstitution experiments, first clearly demonstrated that multivalent interactions are the main driving force for the assembly of BMCs [29]. In the same year, McKnight et al. reported that IDRs can serve as molecular scaffolds, driving the formation of membrane-less organelles via phase separation [30]. These pivotal discoveries collectively revealed the physicochemical principles underlying condensation and propelled systematic studies of diverse cellular condensates, such as stress granules (SGs), nuclear speckles, and Cajal bodies (Table 1).

Table 1 Membraneless organelles formed by phase separation

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The pathological significance of condensation was initially recognized through its association with protein aggregation, which is a key hallmark of many neurodegenerative diseases [41]. In 2015, Hyman and Alberti found that FUS protein undergoes LLPS, which contributes to pathological aggregate formation in neurons of amyotrophic lateral sclerosis (ALS) patients [42]. Subsequent studies further revealed that the phase separation of tau protein facilitates the formation of neurofibrillary tangles in Alzheimer’s disease, and that mutant HTT protein in Huntington’s disease promotes neurotoxic aggregate formation via phase separation. These advances indicate that dysregulation of biomolecular phase separation can convert reversible liquid droplets into irreversible pathogenic aggregates, thereby contributing to the development and progression of neurodegenerative diseases.

As research on biomolecular phase separation progresses, the roles of condensation have expanded well beyond its initial association with pathological protein aggregation. Biomolecular phase separation is now regarded as a dynamic and regulated molecular organizational mechanism that participates in a wide array of biological processes. Among these, transcriptional regulation has emerged as a major focus of BMCs. In 2012, McKnight et al. discovered that the FUS, EWS, and TAF15 proteins contain low-complexity (LC) domains, which are capable of aggregating in vitro to form hydrogel-like structures [30, 43]. While these studies primarily focused on hydrogel formation, they provided important insights into the potential role of biomolecular phase separation in transcriptional regulation [43, 44]. In 2017, Sharp and Young proposed an innovative conceptual model in which super-enhancers (SEs) may use LLPS to cluster large numbers of transcription factors and coactivators at specific genomic regions to activate transcription [45]. In 2018, Young’s team demonstrated through further experiments that coactivators BRD4 and MED1 form liquid-like nuclear puncta via LLPS in SE regions, which enhances the expression of key genes [46]. Recent advances in super-resolution live-cell imaging, cryo-electron microscopy, mass spectrometry and in vivo labeling have greatly expanded knowledge of condensation functions. In addition to transcriptional regulation, biomolecular phase separation has been found to participate in a variety of biological processes, including maintenance of chromatin structure, mRNA translation, signal transduction, and immune responses [47, 48]. The physiological and pathological roles of condensation are equally broad, spanning the neural, cardiovascular, hematopoietic, and reproductive systems, among others, and are closely associated with tumorigenesis.

More importantly, condensation is gradually transitioning from a biological concept to an actionable therapeutic target, representing a new frontier in drug development. Current therapeutic strategies targeting condensation focus on different mechanistic nodes, including directly modulating phase separation processes and utilizing condensation features to design novel drug delivery systems. Biomolecular phase separation research has progressed from basic mechanistic studies to disease association and clinical translation.

Structural basis of biomolecular phase separation

Biomolecular phase separation is a thermodynamically spontaneous process that results from multivalent interactions, including π–π stacking, electrostatic attractions, and hydrogen bonding. The collective effect of these interactions reduces the system’s free energy, ultimately driving phase separation [1, 8, 49, 50]. Although macromolecular aggregation during phase separation appears to increase order and reduce system entropy, recent studies suggest that the release of structured water from hydration shells actually leads to an overall entropy gain, serving as a major contributor for condensation [51]. Meanwhile, multivalent interactions lower the system’s enthalpy. Together, these effects reduce free energy and promote the formation of BMCs. Biological processes such as transcriptional activation and oncogenic signal transduction are frequently associated with multivalent interactions among relevant proteins and the occurrence of condensation within cells. The multivalent interaction capacity of proteins primarily depends on IDRs and multiple folded domains (Fig. 2). IDRs, with their abundance of flexible interaction sites, mediate various weak interactions, while multiple folded domains enable multivalent connections through repeated specific binding. These structural features endow proteins with multivalency, thus providing the structural basis for biomolecular phase separation.

IDR

Biomolecular phase separation can be facilitated by specific features within amino acid sequences, particularly IDRs enriched in LC sequences. These IDRs and LC sequences exhibit significant overlap and are frequently used interchangeably in the literature. IDRs lack stable tertiary structure but are enriched in amino acid residues prone to interactions, including aromatic, charged, and polar side chains [52]. In recent years, Pappu et al. proposed the “sticker-spacer” model to describe the molecular mechanism of IDR-mediated phase separation [53]. In this model, “stickers” represent key residues engaging in multivalent binding via weak interactions, including π-π stacking, cation-π interactions, electrostatic attraction, or hydrogen bonding, that primarily promote condensation. “Spacers” are intervening sequences that do not participate in interactions but enhance protein fluidity and prevent aberrant aggregation. Alterations in sticker composition, spacing, or valency through mutations, truncations, or post-translational modifications (PTMs) can shift condensates from liquid-like states to gel-like or solid-like aggregates [54]. Such aberrant phase transitions contribute to neurodegenerative diseases and cancers [42, 55]. While classical models emphasized the dominant role of specific residues such as aromatic or charged residues, recent studies reveal that non-canonical sequence motifs can also achieve multivalency through cooperative weak interactions under varying sequence contexts and environmental conditions [52, 54, 56,57,58]. This highlights that biomolecular phase separation does not rely on rigid, sequence-specific features but rather exhibits remarkable diversity and flexibility in its driving forces.

Multiple folded domains

Multivalent folded domains constitute another factor driving the multivalency necessary for biomolecular phase separation. These structural units mediate the assembly of condensates through repeated interaction motifs. Classic examples of modular domain motif interactions include the SH3–PRM and SUMO–SIM pairs. In multidomain proteins, multiple repetitive modular domains (RMDs) are linked by short, flexible peptide segments. This arrangement allows individual domains to move flexibly, enabling multivalent interactions that facilitate condensation. In contrast to IDRs, which mainly mediate phase separation through transient, nonspecific, and weak interactions, RMDs achieve multivalency via multiple, specific interactions between domains and their cognate binding motifs. Although these domain–motif interactions are typically of weak to moderate affinity, their multivalent nature allows large numbers of protein molecules to cooperate, maintaining the dynamic equilibrium of condensates [59]. A well-characterized example is the Nephrin/Nck/N-WASP signaling complex. In this system, phosphorylation of Nephrin exposes tyrosine residues, which enables specific recruitment of Nck via its SH2 domains. Meanwhile, the SH3 domains of Nck bind to PRM sequences in N-WASP, establishing a spatially organized network of multivalent interactions [29, 59]. The integration of these modular binding events promotes condensate formation and coordinates signaling processes.

Moreover, the self-association ability of RMDs can significantly enhance their multivalent binding capacity [60, 61]. A typical example is nucleophosmin, whose N-terminal domain forms a pentamer, conferring sufficient multivalency to bind arginine-rich motifs and initiate condensation. Many modular domains such as DIX, PB1, or SAM domains possess specific regions that enable precise homotypic interactions, allowing them to assemble in an ordered fashion into linear or helical supramolecular structures, which markedly increases their multivalent binding capacity [62,63,64].

Regulatory factors of biomolecular phase separation

Intrinsic factors

The intrinsic factors influencing biomolecular phase separation primarily include protein concentration and structural features. Among these, protein structural characteristics can be regulated by factors such as gene mutations, gene fusion, and PTMs.

Protein concentration

When the protein concentration exceeds the critical saturation threshold, weak multivalent interactions can synergistically promote protein aggregation, driving the formation of condensates (Fig. 3a) [65]. This concentration dependence can be captured in phase diagrams, where the binodal curve clearly demarcates homogeneous distribution from phase separation [66]. Under physiological conditions, most endogenous proteins remain soluble as their concentrations fall below this triggering threshold. Thus, the threshold concentration establishes a permissive window for cellular responses to environmental signals: below this level, biophysical stimuli like pH or temperature shifts are generally inadequate to induce condensation [67]. Only significant upregulation or local enrichment of protein expression pushes concentrations into the phase separation range. Furthermore, recent studies have shown that heterogeneous distributions of clusters, rather than fully phase-separated droplets, can form even below the saturation concentration. These subsaturated clusters increase in size as the protein concentration rises, with some encompassing tens to hundreds of molecules. Such observations suggest a progressive shift in molecular organization prior to phase separation [68]. Importantly, persistent overexpression not only promotes phase separation but can also transform dynamic, reversible condensates into pathological, irreversible aggregates [69].

Changes in protein concentration and the resulting phase separation are closely linked to cellular homeostasis. When cells experience stress that leads to a rapid increase in protein synthesis, phase-separated droplets can temporarily sequester excess proteins. This prevents accumulation toxicity and provides cells with flexible, tunable compartmentalization [70]. cells employ phase separation to form membraneless compartments that enable selective molecular partitioning. For instance, DDX4 condensates can utilize electrostatic repulsion to selectively enrich positively charged molecules while excluding negatively charged or neutral species [71].

Protein structure

Gene mutations

Gene mutations primarily affect biomolecular phase separation by altering the multivalent interaction capacity, conformational state, or interaction specificity of the associated proteins they encode (Fig. 3b). Pathogenic gene variants, such as mutations in the FERM domain of NF2, activating mutations in SHP2, and the V225A mutation in APP, can disrupt autoinhibitory mechanisms or enhance binding affinities, increasing multivalent intermolecular interactions and promoting condensation propensity [72,73,74]. Similarly, truncating mutations in the genes encoding tumor suppressors such as APC and p53, or oligomerization-deficient mutations in the RNA helicase DDX24, may result in the loss of critical interaction motifs or IDRs, thereby inhibiting condensates formation [75,76,77]. Additionally, gene mutations that alter LLPS-prone proteins, such as α-synuclein, SOD1, and tau, may not abolish phase separation but rather alter the material properties of the condensates. Such alterations can induce a transition from reversible liquid-like droplets to irreversible amyloid aggregates, promoting the progression of neurodegenerative diseases [78,79,80,81]. Furthermore, mutations in FUS associated with ALS and frontotemporal lobar degeneration can affect its interaction specificity with wild-type protein during LLPS, modulating disease progression [82].

Gene fusion

Oncogenic fusion proteins resulting from chromosomal translocations frequently retain IDRs or prion-like domains (PLDs) derived from their parental proteins, endowing them with phase separation capability (Fig. 3c). Concurrently, these fusion proteins acquire DNA-binding domains, which facilitate precise localization to specific chromatin regions. This chimeric architecture promotes the assembly of condensates at defined DNA regulatory elements, conferring novel functionalities to the fusion proteins. Prototypical oncogenic fusion proteins, FUS-CHOP and EWSR1-FLI1, retain the PLDs characteristic of the FET protein family, and are capable of recruiting chromatin remodeling complexes such as SNF2H or the BAF complex at enhancers [83, 84]. In the FUS-DDIT3 fusion protein, prion-like domains from FUS and DDIT3 engage in heterotypic oligomerization, which facilitates condensate formation within the cell. These condensates subsequently recruit SWI/SNF chromatin remodeling complexes, resulting in alterations of chromatin architecture and transcriptional regulation [85]. Similarly, NUP98 fusion proteins, NUP98–HOXA9 and NUP98–KDM5A, exploit their phenylalanine-glycine repeat-rich IDRs to spontaneously undergo LLPS, establishing transcriptional regulatory hubs at leukemogenic chromatin sites [86, 87]. And this LLPS property is intrinsic to the NUP98 domain and is maintained even in recombinant constructs containing solely the essential FG repeat motif [88].

PTMs

Phosphorylation: Phosphorylation can regulate biomolecular phase separation by altering protein charge distribution, conformational stability and intermolecular interactions, influencing both the formation and functional properties of BMCs (Fig. 3d). For example, phosphorylation within the IDR of HDAC6 enhances its phase separation ability and this PTM provides a molecular basis for its subsequent nuclear functions (Table 2) [89]. Cyclin T1-derived condensates selectively recruit the phosphorylated C-terminal domain of RNA Pol II, creating a phosphorylation-dependent microenvironment that promotes processive transcriptional elongation [90]. Similarly, phosphorylation of p53 at Ser392 augments its LLPS capability and enhances its DNA-binding efficiency within condensates [91]. It is noteworthy that the regulatory effects of phosphorylation on condensation can be bidirectional. For instance, phosphorylation of FOXM1 disrupts the charge landscape of its IDRs, weakening its phase separation propensity and interaction with chromatin, ultimately enhancing tumor immunogenicity [92]. Phosphorylation of CPSF6 inhibits its LLPS capability and shifts poly(A) site selection toward proximal sites, facilitating the production of oncogenic mRNA splice variants. Furthermore, autophosphorylation of CLK2 destabilizes its condensates within nuclear speckles, contributing to the dynamic regulation of spliceosome recruitment under stress conditions [93].

Lactylation: Lactylation, a metabolism-associated PTM, serves as a vital bridge between cellular metabolites and protein function. This modification is facilitated by alanyl-tRNA synthetases AARS1 and AARS2, which act as lactate sensors enabling widespread lysine lactylation within tumor cells. The targets of AARS-mediated lactylation are diverse, encompassing tumor suppressor p53 and the innate immune sensor cGAS. Specifically, lactylation at lysine residues K120 and K139 within the DNA-binding domain of p53 markedly impairs its LLPS ability, compromising its DNA-binding and transcriptional activation activities [94]. Likewise, lactylation of cGAS suppresses its LLPS properties, diminishes its DNA sensing capability and innate immune activation, and promotes tumor- or virus-induced immune evasion [95].

Methylation: Arginine methylation, through the addition of methyl groups to arginine side chains, attenuates cation–π interactions between arginine and aromatic residues, diminishing the strength of multivalent weak interactions between proteins. This modification helps prevent excessive aggregation of BMCs and maintains their homeostasis. Evidence from research on neurological disorders demonstrates that arginine methylation of FUS and hnRNPA2 restricts pathological protein aggregation and protects neuronal function, whereas hypomethylation increases protein multivalent interactions and promotes the transition from liquid-like to solid-like states [96, 97]. Given that FUS exerts critical biological functions in a range of tumor types, it is plausible that its methylation status may similarly modulate condensation, impacting tumor cell behavior [98]. This hypothesis merits further investigation within the context of tumor biology.

Acetylation: Acetylation can both promote biomolecular phase separation by stabilizing proteins and facilitating condensate formation, or inhibit condensation by interfering with critical intermolecular interactions. In autophagy regulation, acetylation of lysine residues in the RB1CC1 N-terminal domain competitively blocks ubiquitin-mediated degradation at these sites. This stabilization promotes the formation of functional condensates, sustaining active autophagic function [99]. Acetylation may also exert detrimental cellular effects by promoting aberrant condensation. For example, in DNA damage repair, acetylation of histone H2A recruits the chromatin reader protein BRD4, which undergoes phase separation at DNA damage sites, forming condensates. These condensates localize to the DNA break ends, competitively hindering the proper recruitment and assembly of repair factors, impairing the repair of DNA double-strand breaks [100]. In antiviral immunity, acetylation demonstrates an inhibitory effect on biomolecular phase separation. Acetylation of IRF3 and IRF7 suppresses their ability to undergo LLPS with DNA. This disruption suppresses type I interferon signaling and attenuates immune responses [101].

Ubiquitination: Ubiquitination can regulate biomolecular phase separation through the assembly of condensates. In neuroblastoma, TRIM37-mediated ubiquitination of PLK4 does not alter its expression level but suppresses the self-assembly of PLK4 into condensates. High levels of this type of ubiquitination impede acentrosomal spindle assembly, resulting in mitotic failure and inhibition of cell proliferation [102]. In contrast, ubiquitination can also promote condensation by enhancing the recruitment of multivalent adaptor proteins. For example, ubiquitination increases the multivalent interaction capacity of PAICS, facilitating its LLPS with UBAP2, a protein containing ubiquitin-binding domains and IDRs, driving purinosome assembly. Abundant purinosome formation enhances de novo purine synthesis pathway flux, to which tumor cells are addicted to sustain their metabolic capacity under stress or high demand, driving tumor progression [103].

Others PTMs: SUMOylation promotes biomolecular phase separation by conjugating SUMO moieties to telomere-binding proteins, which enables multivalent SUMO-SIM interactions and promotes the assembly of PML nuclear bodies. This process enhances telomere clustering and attenuate cellular senescence [104, 105]. During the DNA damage response, PARylation of P-TEFb, specifically at its Cyclin T1 subunit, inhibits phase separation, which leads to suppression of RNA Pol II elongation and promotes the initiation of DNA repair [106]. In leukemia, neddylation can impede the LLPS of the oncoprotein PML/RARα, disrupt the architecture of PML nuclear bodies, and compromise their function, promoting tumor progression [107].

Although these PTMs operate through distinct molecular mechanisms, they primarily influence phase separation by modulating the multivalency or interaction capabilities of substrate proteins. For example, proteins with multiple PTM-binding domains linked by flexible regions can undergo condensation in a manner that is highly sensitive to PTMs [108, 109]. PTMs can be precisely regulated by modulating the activity of specific modifying enzymes, so it is possible to selectively manipulate cellular processes related to BMCs. Accordingly, PTMs not only help to elucidate the molecular basis of biomolecular phase separation regulation, but also offer novel strategies and potential therapeutic targets for the intervention and treatment of related diseases. Condensation alterations have been observed following the use of several clinically established drugs, such as kinase and PARP inhibitors [110, 111]. However, whether the modulation of condensation constitutes a primary mechanism underlying their therapeutic effects remains an open question.

Table 2 The influence of different PTMs on phase separation

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Multicomponent interactions

Intracellular BMCs are typically composed of multiple proteins and RNAs, representing classical examples of multicomponent systems. In simplified single-component models, it is generally assumed that the saturation concentration in the dilute phase remains constant. However, recent studies have demonstrated that this assumption does not hold true in living cells, where the highly multicomponent nature of condensates leads to more complex phase behavior (Fig. 3e). For instance, the saturation concentration of endogenous condensates, such as the nucleolus, has been reported to vary with the concentrations of other components, indicating that heterotypic interactions can reshape phase boundaries [112]. Further studies have revealed that the phase behavior of a given protein is often influenced by its interaction partners. For example, although FUS can undergo spontaneous LLPS in vitro, its condensation can be suppressed by the nuclear transport receptor karyopherin-β2 through multivalent interactions with distinct regions of FUS [113]. Similarly, in neuronal synapses, the LLPS of gephyrin is regulated not only by its intrinsic structural features and concentration but also by precise interactions with neurotransmitter receptors and other components within the condensate, through diverse binding modes and affinities [114]. Such heterogeneous regulatory mechanisms are further supported by the stickers-and-spacers model. Studies have shown that ligands, although unable to undergo phase separation on their own, can still modulate scaffold protein LLPS through their valency, binding sites on scaffolds, and binding affinities [115]. These findings underscore the complexity and regulatory significance of multicomponent interactions in controlling phase behavior of BMCs.

Extrinsic factors

Temperature

The effects of temperature on biomolecular phase separation can be categorized into two distinct response patterns known as upper critical solution temperature (UCST) and lower critical solution temperature (LCST) (Fig. 3f). In the UCST-type pattern, phase separation is favored at lower temperatures, where cooling enhances intermolecular interactions such as hydrogen bonding, electrostatic interactions and aromatic stacking. This behavior is particularly prevalent in proteins enriched with IDRs, such as FUS and Swc5 [116, 117]. Conversely, LCST-type behavior is characterized by phase separation that occurs at elevated temperatures. Heating disrupts the structured hydration shell around hydrophobic residues, increasing system entropy and strengthening hydrophobic interactions, which promote protein aggregation. A canonical example of LCST behavior is observed in elastin-like polypeptides (ELPs), particularly those containing the repetitive VPGVG motif. Studies have shown that as the temperature increases, ELPs undergo gradual conformational changes, including exposure of hydrophobic valine side chains and partial ordering of the peptide backbone. These changes disrupt the structured hydration shell surrounding hydrophobic residues, leading to the release of bound water molecules. Simultaneously, enhanced hydrophobic interactions promote inter-peptide aggregation, thereby promoting phase separation. The process reflects a shift in the balance between peptide-water and peptide-peptide interactions, governed by temperature-induced structural rearrangements [118]. Notably, condensation regulated by temperature is typically reversible, as condensates can rapidly assemble or disassemble in response to temperature fluctuations. However, within the cellular environment, the assembly of membraneless organelles such as the nucleolus is orchestrated by both thermodynamic phase separation and active assembly mechanisms. These active processes can modulate or even override the intrinsic reversibility of BMCs. This highlights the complexity of condensate formation in vivo and underscores the importance of integrated approaches for understanding their dynamic behavior [119].

pH

pH frequently regulates biomolecular phase separation by altering the charge distribution and hydrophobicity of proteins (Fig. 3g) [120, 121]. Under acidic conditions, increased protein protonation leads to higher positive charges and enhanced electrostatic interactions, promoting condensation. For instance, exposure to low pH induces the folding and aggregation of the yeast prion protein Sup35 and prothymosin-α [122, 123]. Additionally, low pH can increase protein hydrophobicity, which further promotes condensation [124]. At high pH, proteins are less protonated and electrostatic interactions are weakened. As a result, protein aggregation is inhibited and solubility increases, which is exemplified by the suppression of LLPS in Pub1 condensates under alkaline conditions [125].

Salt

Salt can modulate electrostatic interactions among proteins (Fig. 3h). Generally, low salt concentrations strengthen intermolecular attractions and facilitate biomolecular phase separation, while high salt levels shield charges and suppress condensate formation. This trend has been consistently demonstrated in various systems, including Ddx4, LAF-1, and FUS [126,127,128]. Interestingly, certain systems display reentrant phase behavior, where condensation is observed exclusively at intermediate salt concentrations. In these cases, insufficient salt cannot overcome intermolecular repulsion, whereas excessive salt disrupts the interactions required for condensation [129]. Moreover, multivalent cations, such as La ³+ and Mg ²+, further influence the viscosity and dynamic properties of condensate droplets [130]. Under stress conditions, alterations in the ionic environment also affect protein localization and toxicity, as reported for FUS and TDP-43 in ALS models [131, 132]. These findings underscore that the regulation of biomolecular phase separation by salt does not follow a simple linear relationship, but rather a complex phenomenon shaped by the intricate interplay among ion concentration, valency, and the molecular environment.

ATP

Adenosine triphosphate (ATP) is amphiphilic in nature, due to its charged triphosphate group and an aromatic ring, which enables it to interact with both water molecules and basic amino acid residues [133] (Fig. 3i). Based on these molecular features, ATP engages in multivalent electrostatic interactions with Arg/Lys residues located in the IDRs of the FUS, enhancing protein hydration and intermolecular association to promote LLPS. However, this process demonstrates a biphasic regulatory pattern, which excessive ATP can saturate the interaction sites, destabilize the condensates, and suppress LLPS [134,135,136,137,138,139]. A similar biphasic regulatory effect has also been observed in other IDR-containing proteins, such as TDP-43 [140]. In addition, ATP-mediated regulation of condensation is further modulated by ionic conditions, particularly Mg ²+, which can chelate ATP and alter regulatory efficacy [141, 142].

Stress

Environmental stresses such as heat shock, oxidative stress, energy deprivation, and hypoxia pose threats to cellular homeostasis. In response, cells form SGs, membrane-less organelles assembled via phase separation, as part of the adaptive mechanisms (Fig. 3j). SG assembly has been shown to involve the inhibition of translation initiation, which promotes multivalent interactions between untranslated mRNAs and RBPs [143, 144]. Through this process, SGs transiently sequester mRNAs and proteins, reducing energy consumption and enhancing cellular survival. Under stress conditions, the assembly and disassembly dynamics of SGs are finely tuned by multiple factors including protein solubility, RNA load, and cellular ATP levels [145,146,147]. Acute SGs are generally protective, whereas chronic SGs can persist and contribute to pathological outcomes [148]. For example, under oxidative stress, TDP-43 can undergo intra-condensate demixing within SGs, transforming into stable pathogenic aggregates closely associated with neurodegenerative diseases [149]. In cancer, dysregulated SG assembly may facilitate cellular adaptation to stress and contribute to therapy resistance. For example, in hepatocellular carcinoma, RIOK1-mediated LLPS leads to SG formation that sequesters PTEN mRNA, suppresses its translation, and promotes metabolic reprogramming, ultimately contributing to resistance against tyrosine kinase inhibitors [150]. In non-small cell lung cancer, cisplatin induces the formation of noncanonical SG–like structures that are associated with cancer cell death. In contrast, cisplatin-resistant cells retain canonical SGs whose disruption has been shown to restore drug sensitivity [151]. Similarly, a clinically identified D336G mutation of UTX increases its cytoplasmic accumulation and stabilizes SGs, thereby promoting cell proliferation and tumorigenesis in a G3BP1-dependent manner [152]. Recent studies have also shown that SGs can mediate intercellular signaling within the tumor microenvironment by packaging and delivering specific mRNAs [153]. Additionally, SGs can rapidly assemble as plugs at sites of endomembrane damage, facilitating the efficient repair of damaged endolysosomes [154]. Altogether, stress responses mediated by phase separation are increasingly recognized not merely as defense mechanisms but as important regulatory processes involved in translational control, cellular homeostasis, and membrane repair. As such, SGs and their underlying phase separation behavior have emerged as promising therapeutic targets in cancer, neurodegenerative disorders, and metabolic diseases.

Metabolites

Metabolic products can regulate biomolecular phase separation, influencing cellular stress responses and signal transduction (Fig. 3k). Glycogen accumulation promotes its own LLPS and suppresses the Hippo signaling pathway, driving liver tumorigenesis [155]. Lactate is utilized in the lactylation of key proteins to regulate LLPS [95]. Amino acids, such as glutamine, modulate condensation to affect the expression of metabolic enzymes, enabling tumor cells to adapt to nutrient deprivation [95, 156]. Sorbitol, a mechano-sensitive metabolite, promotes LLPS as a natural crowding agent, linking external mechanical cues to intracellular functions and helping cellular adaptation to mechanical stress [157]. Insulin promotes IRS1 LLPS to mediate precise regulation of the insulin signaling pathway, whereas in insulin-resistant states, impaired formation of these condensates leads to defects in signal transduction [158].

Molecular chaperones

Molecular chaperones are mainly responsible for protein folding, disaggregation, and degradation, maintaining protein homeostasis and quality within the cell (Fig. 3l). Although some chaperones do not directly undergo phase separation, they regulate the client proteins that enable LLPS through various mechanisms. For example, classical molecular chaperones such as Hsp70 and Hsp90 modulate protein folding and disaggregation, safeguarding the dynamic equilibrium of condensates and ensuring cellular function [159]. Small heat shock proteins, such as Hsp27 and Hsp22, can interact with FUS and tau to maintain the liquid state of condensates and prevent amyloid fibril formation [146, 160]. The molecular chaperone peptidyl prolyl isomerase A can be recruited into tau condensates, where it triggers their dissolution [161]. Additionally, Hsp40 family proteins can utilize their glycine/phenylalanine-rich region to partition into BMCs and cophase separated with FUS or SGs to promote the formation and stability of these assemblies [162, 163].

Nucleic acids

Nucleic acids modulate biomolecular phase separation through multiple mechanisms, including sequence characteristics, structural configurations, chemical modifications, and concentration-dependent effects (Fig. 3m). Repeat-expanded RNAs undergo age-dependent aggregation transitions within multi-component condensates such as SGs, resulting in the formation of condensates with an RNA-rich solid core surrounded by an RNA-depleted fluid shell. Such aberrant aggregation has been implicated in numerous neurological disorders [164].The secondary structure of mRNA governs its propensity for self-association and dictates whether it is incorporated into liquid compartments [165]. Long non-coding RNA (lncRNA), exemplified by NEAT1, contains functionally redundant subdomains that preferentially bind NONO and SFPQ, facilitating LLPS and constructing nuclear paraspeckles [166]. Additionally, chemical modifications of RNA, such as N6-methyladenosine (m6A), could enhance the multivalent scaffolding ability of mRNAs, promote the LLPS of YTHDF, and support the formation of SGs [167, 168]. RNA further exerts concentration-dependent, bidirectional regulation of condensation. At low RNA-to-protein ratios, droplet formation is promoted, whereas high ratios saturate protein binding sites and suppress or destabilize condensates. This phenomenon is widely observed in FUS and TDP43 condensates [55, 169]. In addition to regulating condensate assembly, recent studies have revealed that RNA also tunes the biomolecular density of intracellular condensates. Acting as long scaffolds, RNA molecules recruit RBPs and form mesh-like transient molecular networks, mitigating the requirement for tight physical contacts between proteins and decreasing the condensate density. This mechanism promotes the formation of low-density condensates, such as nuclear speckles and SGs, which exhibit increased permeability [170]. Similarly, DNA can mediate LLPS via its repetitive sequences in cooperation with heterochromatin proteins such as HP1α, contributing to the formation of nuclear heterochromatin domains and the maintenance of gene silencing and genome integrity [14, 171, 172].

Biomolecular phase separation regulates the hallmarks of cancer

Biomolecular phase separation regulates tumor proliferation

Biomolecular phase separation regulates the stability and expression levels of proliferation-associated molecules, influencing tumor cell proliferation (Fig. 4). For example, in breast cancer cells, lncRNA CD2BP2-DT promotes YBX1 LLPS, forming protective condensates. These condensates enhance the stability of CDK1 mRNA, which encodes a cyclin-dependent kinase critical for cell cycle progression, promoting cell proliferation [173]. In hepatocellular carcinoma, FOXM1 forms LLPS-dependent complexes with ASPM, co-occupying multiple genes to synergistically enhance their expression and accelerate malignancy [174]. In addition, FOXM1 can undergo phase separation with specific DNA elements, remodeling the nuclear transcriptional environment, sustaining chromatin accessibility and S landscapes that are critical for tumor cell proliferation [92]. Beyond transcriptional control, condensation regulates RNA processing. Increased condensation of CPSF6 promotes preferential usage of distal poly(A) sites during alternative polyadenylation (APA) [93]. Similarly, in colorectal cancer, PABPN1 LLPS suppresses proximal poly(A) site selection [175]. Tumor cells disrupt this repressive regulation through phosphorylation of CPSF6’s arginine/serine-like domain or competitive binding to PABPN1’s glutamic acid-proline domain, impairing their respective condensation. This shifts APA toward proximal sites, generating shortened 3’UTRs in proliferation-related mRNAs that evade miRNA silencing, thereby promoting proliferation. It is noteworthy that not only the occurrence but also the degree of condensation is crucial. For instance, in acute myeloid leukemia (AML), only moderate LLPS between YY1 and HDAC1/3 can activate METTL3 expression and promote cell proliferation, whereas excessive LLPS exerts the opposite effect [176]. Recent studies have shown that cancer-associated mutations in ENL promote the formation of submicron-sized condensates at native genomic targets, which activate oncogenic gene expression. However, overexpression of ENL mutants leads to the formation of large, non-functional condensates that fail to activate transcription. The underlying biophysical mechanisms that govern this transition in response to different expression levels remain to be elucidated [177].

Biomolecular phase separation regulates tumor metastasis

Tumor metastasis depends on a range of acquired traits, including enhanced invasive and migratory capacities, induction of epithelial-mesenchymal transition (EMT), and cytoskeletal remodeling [178]. Recent advances have shown that biomolecular phase separation modulates these metastatic traits in a context-dependent manner, exerting either pro- or anti-metastatic effects. Pro-metastatic condensation events have been reported in several settings. For example, the splicing factor SRSF9 generates truncated oncogenic isoforms via phase separation, promoting the metastatic potential in oral cancer [179]. CKAP4, acting as an intracellular mechanosensor for mechanical stress, forms numerous micron-scale puncta through condensation to remodel the cytoskeleton and facilitate cell spreading [180]. In addition, DDX21 undergoes LLPS to concentrate at the MCM5 genomic locus, activating its transcription and subsequently initiating EMT-driven metastasis in colorectal cancer [181]. Conversely, biomolecular phase separation can also suppress metastasis in certain contexts. In hepatocellular carcinoma, nuclear YBX1 forms LLPS-driven condensates that accelerate TPM4 mRNA decay, inhibiting cytoskeletal remodeling and metastatic dissemination [182]. Similarly, SQSTM1 and NBR1 assemble into intracellular liquid droplets that promote RAC1 degradation and ultimately inhibit tumor cell motility and metastatic potential [183].

Biomolecular phase separation regulates tumor cell death

Cell death is a natural physiological processes, however, tumor cells frequently evade this fate through diverse strategies such as enhancing autophagy or forming SGs [184]. For example, phosphorylation of HNRNPH1 at tyrosine 210 promotes its LLPS, which in turn promotes alternative splicing of NBR1, activating autophagy and inhibiting apoptosis in colorectal cancer cells. This mechanism supports tumor cell survival under adverse conditions [185]. Similarly, in gastric cancer, phase separation of ATG4B enhances autophagic flux, contributing to resistance against anoikis [186]. In addition, SGs, as classic LLPS-derived BMCs, sequester pro-apoptotic proteins under cellular stress, promoting cell survival. However, impairment of SG clearance leads to their abnormal accumulation, which can instead exacerbate apoptosis [187].On the other hand, biomolecular phase separation can also promote cell death under certain circumstances. For instance, binding of BAX protein to the voltage-dependent anion channel on the mitochondrial membrane is a pivotal step in mitochondrial outer membrane permeabilization and apoptosis initiation, a process that can be competitively inhibited by hexokinase (HK). Tau-441 undergoes LLPS to form condensates that recruit HK, decreasing the amount of free cytosolic HK and reducing its competition with BAX for VDAC I binding, ultimately promoting apoptosis [188]. Furthermore, the Smad2/3/4 complex can form nuclear condensates via LLPS, which enhances its association with the TAT promoter, upregulates its expression, and directly activates downstream apoptotic pathways, increasing apoptosis in hepatoma cells [189]. Together, these findings broaden our understanding of cell death regulation. Further research is needed to investigate the role of biomolecular phase separation in regulating other forms of cell death, such as pyroptosis, ferroptosis, and cuproptosis.

Biomolecular phase separation regulates the evasion of growth suppressors in tumor cells

Tumor development depends not only on the activation of growth-promoting signals but also on the effective evasion of growth-suppressive cues. Cancer cells often suppress classical tumor suppressors such as p53 and SPOP, thereby undermining intrinsic growth control and promoting oncogenesis [190, 191]. P53 is one of the most well-characterized tumor suppressors, and its proper function has been shown to partially rely on phase separation. Aberrant LLPS behavior may compromise p53 activity and even confer oncogenic properties, which is a topic that will be discussed in detail in Sect. 6.3.3. Similarly, SPOP, a cullin-3-RING ubiquitin ligase substrate adaptor, functions as a tumor suppressor whose activity is also regulated by LLPS. Wild-type SPOP self-associates into higher-order oligomers through its BTB and BACK domains, enabling multivalent interactions with its substrates. These multivalent interactions drive the co-phase separation of SPOP and its substrates into condensates within nuclear speckles, facilitating their ubiquitination and subsequent degradation [192,193,194]. Interestingly, researchers have found that certain cancer-associated SPOP mutations, while impairing its oligomerization and substrate binding, paradoxically enhance phase separation with the substrate DAXX and increase polyubiquitination efficiency [195]. These findings suggest that phase-separation-mediated regulation of tumor suppressors may be more nuanced than previously appreciated.

Biomolecular phase separation regulates tumor angiogenesis

Tumor angiogenesis refers to the process of new blood vessels are formed within and around tumors, providing essential nutrients and oxygen to sustain the rapid proliferation of cancer cells. Biomolecular phase separation plays a critical role in this process by regulating the distribution and function of key proteins within endothelial cells (ECs), influencing vascular development, maturation, and pathological neovascularization. For example, the subcellular localization of Kindlin-2 is altered under different shear stress conditions, and LLPS of Kindlin-2 in ECs is essential for proper focal adhesion assembly, maturation, and junction formation. Oscillatory shear stress induces PRMT5-mediated arginine hypermethylation, which inhibits Kindlin-2 LLPS, impairing focal adhesion assembly and junction maturation, ultimately leading to increased vascular permeability [196]. In addition, point mutations in the DEAD-box helicase DDX24 are closely associated with vascular abnormalities such as multi-organ venous and lymphatic defect (MOVLD) syndrome. DDX24 is primarily localized in the nucleolus, where it maintains nucleolar homeostasis through LLPS-mediated interactions with nucleophosmin. Mutant DDX24 exhibits reduced nucleolar localization in tissues from MOVLD patients and in cultured endothelial cells. This mislocalization leads to abnormal nucleolar morphology and impaired ribosome biogenesis, as well as increased endothelial cell migration and tube formation. These findings reveal a molecular mechanism by which DDX24 regulates vascular abnormalities through modulation of NPM1 phase separation [77]. Pharmacological studies have shown that the LLPS inhibitor 1,6-hexanediol (1,6-HD) significantly suppresses angiogenesis and endothelial network formation in various in vivo and in vitro models. Mechanistically, 1,6-HD downregulates the transcription of cell cycle-related genes such as cyclin A1, inhibiting endothelial cell migration, proliferation, and growth [197]. Furthermore, DDR1 undergoes LLPS in response to matrix stiffness or collagen stimulation and co-condenses with LATS1, leading to LATS1 inactivation and subsequent activation of the YAP signaling pathway. This process enables ECs to respond to extracellular matrix remodeling and regulates angiogenesis [198]. Collectively, these findings highlight biomolecular phase separation as a fundamental molecular mechanism underlying tumor angiogenesis and suggest novel therapeutic targets for anti-angiogenic cancer therapy.

Biomolecular phase separation supports the unlimited replicative potential of tumor cells

Certain cancer cells achieve replicative immortality by leveraging LLPS in the alternative lengthening of telomeres (ALT) pathway. Loss of BRCA2 leads to abnormal stabilization of telomeric G-quadruplexes, which promotes the accumulation of telomeric repeat-containing RNA R-loops at telomeres. These R-loops serve as multivalent molecular platforms that contribute to the LLPS of telomere-associated proteins, such as PML and DNA repair factors, resulting in the assembly of ALT-associated promyelocytic leukemia bodies (APBs) [199]. The liquid nature of APBs not only facilitates telomere clustering but also provides an optimal microenvironment for telomere extension reactions, including break-induced replication [104, 200]. In the ALT pathway, SUMOylation of critical proteins in APBs, such as TRF2, enhances the recruitment of DNA repair factors and supports continued telomere elongation [201]. Collectively, LLPS-driven APB assembly and telomere recombination provide a molecular basis for unlimited telomere extension and sustained proliferation in cancer cells lacking telomerase, highlighting biomolecular phase separation as a critical mechanism and potential therapeutic target in ALT-positive tumors.

Biomolecular phase separation regulates tumor cell phenotypic plasticity

The acquisition of phenotypic plasticity enables tumor cells to undergo dedifferentiation, blocked terminal differentiation, and transdifferentiation, enabling adaptation to various microenvironmental changes and stresses [202]. Recent studies have demonstrated that, in arsenic-induced skin damage, YTHDF2 undergoes LLPS upon recognition of m⁶A-modified PTEN mRNA, thereby suppressing PTEN translation and activating the AKT pathway, which promotes the malignant transformation of keratinocytes [203]. In hepatocellular carcinoma, EEF1E1 has been identified as a phase-separating protein involved in tumor stemness and DNA damage repair. Inhibition of EEF1E1 LLPS partially reverses its tumor-promoting effects and leads to downregulation of stemness-related markers such as CD133 [204].

Biomolecular phase separation regulates tumor energy metabolism

To sustain and promote cancer progression, tumor cells need to reprogram their metabolism and energy utilization. For instance, the transcription factor FOXK1 forms nuclear condensates via condensation in renal tubular epithelial cells, thereby enhancing the expression of glycolysis-related genes and promoting a high-glycolytic state [205]. Under glucose deprivation, IGF2BP3 assembles with HK2 mRNA into SGs through LLPS, stabilizing HK2 expression and maintaining glycolytic capacity [206]. In addition, the lncRNA GIRGL promotes the LLPS-mediated interaction between CAPRIN1 and GLS1 mRNA, repressing glutaminase translation and helping cells adapt to glutamine deprivation [156]. During energy exhaustion, K63-linked ubiquitination of HAX1 enhances its condensation, promoting P-body formation. This leads to a global inhibition of protein synthesis, shifting cellular metabolism from a high- to low-energy state. However, this suppression is not uniform. Instead, it represents a conserved eukaryotic mechanism of translational remodeling that selectively regulates which mRNAs are repressed, stored, or translated. This selective control enables cells to prioritize the synthesis of survival-related proteins under energy-limiting conditions [207]. Structural proteins, such as CAMSAP2, and peroxisomal membrane proteins Pex13/Pex14, can also regulate microtubule and peroxisome assembly via LLPS, enhancing lipid metabolism and intracellular transport [208, 209]. Collectively, these studies indicate that biomolecular phase separation confers high metabolic flexibility and adaptability to cells by coordinately regulating multiple metabolic and stress response pathways.

Biomolecular phase separation regulates tumor immune destruction

Immune evasion promotes tumor progression and continues to pose a significant barrier to successful immunotherapy. In T cell biology, biomolecular phase separation facilitates the formation of dynamic signaling condensates, which are essential for antigen recognition. Key adaptor proteins such as LAT, GRB2, and SOS1 form microclusters at the plasma membrane via LLPS, selectively recruiting kinases to amplify T cell receptor signaling [13, 210]. Interestingly, it is these LLPS-driven microclusters that are responsible for the exquisitely sensitive, all-or-none nature of T cell antigen recognition. Furthermore, LAT condensates are molecularly linked to the actin cytoskeleton through NCK and WASP, which directs the movement of signaling complexes toward the immunological synapse [211]. Tumor cells are thought to disrupt this process by inhibiting LAT phosphorylation, promoting its endocytosis and ubiquitin-mediated degradation, thereby lowering its local concentration below the threshold necessary for phase separation. This impairs the formation of TCR signaling condensates and ultimately attenuates T cell effector functions [212, 213]. In addition to disrupting condensation in immune effector cells, tumor cells can actively exploit phase separation to construct immunosuppressive regulatory hubs. For example, the histone acetyltransferase KAT8 forms nuclear condensates with the transcription factor IRF1 via LLPS, enhancing the transcription of the immune checkpoint molecule PD-L1 and promoting tumor immune evasion [214]. On the other hand, biomolecular phase separation can also help suppress immune escape. For instance, the m⁶A reader YTHDF3 leverages phase separation to recruit the RNA helicase DDX6, facilitating degradation of its target HSPA13 mRNA. This downregulates the immune checkpoint PD-L1 and suppresses immune evasion in clear cell renal cell carcinoma [215].

The role of condensation in chimeric antigen receptor (CAR)-T cell signaling and functional maintenance is also significant. Although CAR-T cells can initiate signaling through LLPS-like clustering and partially bypass LAT dependency, the resulting microclusters often fail to fully integrate into mature central supramolecular activation clusters. As a result, CAR-T cells are more prone to spatial disorganization within the immunosuppressive tumor microenvironment, leading to suboptimal and transient activation [216]. In the future, modulating or optimizing biomolecular phase separation processes in CAR-T cells may enhance their signal integration and antitumor efficacy. Moreover, recent studies have shown that tumor cells can evade immune surveillance by rewiring innate immune signaling pathways such as the cGAS–STING axis, a mechanism that will be discussed in detail later.

Biomolecular phase separation regulates tumor epigenetic reprogramming

Biomolecular phase separation has recently been shown to play a widespread role in epigenetic regulation and chromatin reprogramming, and is closely associated with tumorigenesis, cell fate decisions, and genome stability. Condensation promotes the assembly of chromatin regulators, transcription factors, and related enzymes into liquid condensates, thereby profoundly influencing chromatin architecture and gene expression states. In triple-negative breast cancer, phosphorylated HDAC6 forms nuclear condensates via LLPS, thereby altering chromatin accessibility, reprogramming histone acetylation, and disrupting transcription. This mechanism highlights the critical role of biomolecular phase separation in epigenetic reorganization and suggests that targeting such condensates may offer a promising therapeutic strategy against cancer [89]. On the other hand, KDM6A, a histone demethylase, mediates LLPS through its IDR, recruiting and activating histone methyltransferases such as MLL4. This process regulates genome-wide epigenetic modifications and chromatin interactions, while LLPS-disrupting mutations in KDM6A abrogate its tumor-suppressive activity [217]. In addition, heterochromatin-associated proteins such as MeCP2 and IRTKS contribute to heterochromatin assembly and maintenance through condensation, affecting genome accessibility and cellular senescence [218, 219]. In cancer research, condensation of chromatin remodeling factors such as SS18-SSX and ARID1A has been shown to be closely associated with malignant transformation and dysregulated gene expression programs [220, 221]. In acute promyelocytic leukemia, neddylation disrupts the PML/RARα fusion protein LLPS, disassembling nuclear bodies and accelerating leukemogenesis [107].

Molecular mechanisms of biomolecular phase separation

Gene transcription regulation

The processes of transcription initiation, elongation, splicing, and termination are critically dependent on the spatially organized enrichment and dissociation of RNA Pol II and its associated factors within specific nuclear structures. Numerous proteins containing IDRs, such as transcription factors, coactivators MED1, BRD4, and RNA Pol II itself, can form transcriptional condensates through condensation at regulatory genomic loci, such as SEs. Weak, multivalent interactions mediated by IDRs allow these molecules to create reversible, locally concentrated environments, which in turn enhance the efficiency and specificity of gene transcription [46, 222, 223]. The phosphorylation state of the C-terminal domain of RNA Pol II is crucial for its dynamic redistribution among different condensates. Hypophosphorylation favors its recruitment into initiation condensates, whereas hyperphosphorylation facilitates its transition to splicing condensates, orchestrating the orderly progression of transcription [224]. Accumulating evidence indicates that tumor cells are highly dependent on the persistent overexpression of SE-driven oncogenes, which is essential for maintaining their malignant phenotypes [225, 226]. For example, coactivators such as BRD4 and MED1 can form LLPS-driven condensates at SE regions, recruiting large quantities of RNA Pol II and transcription factor, markedly enhancing oncogene expression [46, 227]. In leukemia, the NUP98-HOXA9 fusion protein, which is rich in IDRs, can utilize condensation to promote broad and dense binding across the regulatory regions of multiple oncogenes, displaying a SE-like binding pattern and driving oncogene transcription [228]. Similarly, oncogenic transcription factors including YAP/TAZ and FOXM1 can form nuclear condensates through condensation, modulating the expression of downstream oncogenes (Fig. 5a) [92, 229]. Moreover, epigenetic regulators such as LSD1 can co-localize with BRD4 at SE-associated LLPS structures, collaboratively regulating oncogene expression. Studies have demonstrated that bromodomain and extra-terminal domain inhibitors can disrupt the formation of LLPS-driven transcriptional condensates at SEs by blocking the interaction between BRD4 and acetylated histones, thereby suppressing oncogene overexpression. When used in combination with LSD1 inhibitors, this strategy synergistically impairs tumor-specific SE activity and significantly inhibits the growth of prostate cancer [230].

In addition, RNA molecules act as molecular scaffolds that cooperate with proteins to support and regulate the assembly and disassembly of biomolecular phase separation, further increasing the complexity of transcriptional regulation (Fig. 5b) [231, 232]. For instance, the lncRNA DIGIT can promote the formation of BRD3 condensates at enhancers related to endodermal differentiation, modulating the expression of developmentally relevant transcription factors [232]. Further elucidation of condensation and its associated RNA regulatory mechanisms provide theoretical support and prospects for developing novel cancer-targeted therapies [233, 234].

However, it is important to note that the mechanistic role of biomolecular phase separation in transcriptional regulation remains controversial. Some studies suggest that while the multivalent interaction capability of transcription factor activation domains (ADs) correlates with transcriptional activation strength, the actual formation of droplet-like TF condensates has a neutral or even inhibitory effect on transcriptional activation. Experimental evidence indicates that AD-mediated multivalent interactions mainly enhance transcription by prolonging the chromatin residence time of transcription factors and promoting coactivator recruitment, rather than strictly relying on the occurrence of condensation [235]. Therefore, the precise mechanisms and physiological significance of biomolecular phase separation in transcriptional regulation require further systematic investigation and validation in future studies.

Maintain genome stability

Biomolecular phase separation is fundamentally important for preserving genomic stability, primarily through its involvement in DNA damage repair, chromatin compartmentalization, and the dynamic remodeling of chromatin architecture. Firstly, during the DNA damage response, condensation accelerates the rapid accumulation and organized assembly of repair proteins. For example, after DNA double-strand breaks(DSB) occur, proteins such as the MRE11-RAD50-NBS1 complex, FUS, 53BP1, and RNF168 utilize their IDRs to form phase-separated condensates at damage sites, which is efficiently to recruit additional repair factors and accelerate the propagation of damage signaling and repair processes [236,237,238]. In addition, non-coding RNAs can further boost DNA repair efficiency by mediating LLPS of repair proteins [98, 239].

Biomolecular phase separation also profoundly influences DNA repair capacity and cellular sensitivity to therapy. For instance, when SUMOylation-induced LLPS of RNF168 occurs, its recruitment to DNA damage sites is restricted, impairing nonhomologous DNA end joining repair efficiency (Fig. 5c) [239]. In various cancer cells, proteins such as KAT6A, USP49, and NONO display abnormally enhanced condensation during the DNA damage response, significantly enhancing DNA repair, fostering drug resistance, and supporting tumor cell survival [240,241,242]. Specifically, KAT6A, with the assistance of APEX1, uses its IDR to promote LLPS at DNA damage sites, forming droplet-like complexes that reduce PARP1 trapping at damage loci. This lowers the cytotoxicity of PARP inhibitors and promotes the emergence of drug resistance [240]. USP49, upon phosphorylation by ATM kinase following radiotherapy, exhibits increased LLPS capacity, assembling highly concentrated repair condensates at DSB sites that efficiently recruit homologous recombination factors such as RPA70 and Rad51, enhancing homologous recombination repair and conferring radiotherapy resistance [241]. Similarly, NONO relies on IDR-mediated LLPS to form nuclear droplets after DNA damage, recruiting and organizing DNA repair protein complexes, which promotes DNA repair and increases tumor cell resistance to radiotherapy [242]. Collectively, these cases indicate that aberrant enhancement of LLPS in tumor-associated proteins contributes to more efficient DNA repair in cancer cells, enables escape from therapy-induced cell death, and increases the risk of drug resistance and tumor progression.

Biomolecular phase separation also participates in the spatial compartmentalization of chromatin. In eukaryotic cells, the histone tails of chromatin can spontaneously undergo LLPS, forming dense and reversible droplet structures that provide a physical basis for chromatin compartmentalization [47]. For instance, heterochromatin marked by H3K9me2/3 can undergo phase separation through the multivalent interactions of HP1 proteins, resulting in high-density chromatin aggregates that shield transposons and repetitive sequences, safeguarding genome stability [14, 171, 243]. LLPS-derived condensates can also cluster functionally related chromatin regions together while excluding unrelated segments, thereby mediating spatial reorganization of chromatin and regulating gene expression [244, 245]. In breast cancer, disruption of phosphorylated HDAC6-mediated LLPS induces extensive chromatin architectural reorganization, including new chromatin loop and topologically associating domain boundary formation. These structural alterations reshape functional chromatin compartments and ultimately influence gene regulation (Fig. 5d) [89].

Importantly, dysregulated biomolecular phase separation can also lead to chromatin disorganization, aberrant gene expression, and tumorigenesis. In synovial sarcoma, LLPS of the SS18-SSX fusion protein facilitates the aberrant enrichment of chromatin remodelers such as BRG1, driving the abnormal activation of related genes and tumor transformation [220].

As a critical molecular aggregation mechanism, biomolecular phase separation underlies the development, drug resistance, and progression of tumors. Therefore, elucidating the specific roles and regulatory mechanisms of condensation in maintaining genome stability will provide valuable theoretical insights for understanding cell fate and for developing innovative precision medicine strategies [14, 89, 92, 239, 246, 247].

Regulation of signal transduction

Signal fidelity depends not only on molecular composition, but also on precise spatial and temporal regulation, with condensation serving as critical mediators of this process.

Hippo pathway

The principal effector TAZ in the Hippo signaling pathway not only forms nuclear condensates via LLPS, but also selectively recruits essential cofactors, including the DNA-binding partner TEAD4, transcriptional coactivators BRD4 and MED1, and the transcription elongation factor CDK9, to establish a highly efficient transcriptional activation platform (Fig. 6a). The phase-separation ability of TAZ relies mainly on its coiled-coil (CC) domain and is negatively regulated by upstream Hippo kinases such as LATS through phosphorylation. Either deletion of the CC domain or substitution with the homologous domain from YAP markedly impairs the LLPS capacity of TAZ and consequently attenuates the transcriptional activation of its target genes [229]. Notably, the formation of TAZ condensates requires not only its own structural features but also support from the nuclear matrix protein NONO. NONO facilitates both the phase separation and transcriptional activity of TAZ, and further modulates its interactions with TEAD, RNA Pol II, and enhancers. Clinical studies have shown that co-upregulation of NONO and TAZ in glioblastoma predicts poor prognosis, while NONO knockdown significantly suppresses TAZ-driven tumorigenesis [248]. Additionally, the protein FUS interacts with the CC domain of TAZ via its LC domains, maintaining the liquidity and dynamic properties of TAZ condensates, which are critical for robust transcriptional activity. Loss of FUS leads to a transition of TAZ condensates toward a gel-like or solid state, reducing target gene expression and pro-tumorigenic activity [249]. Upstream Hippo kinases are also subject to LLPS-mediated regulation. For example, LATS2 utilizes its proline-rich motif (PRM) to undergo LLPS, assembling into dynamic signalosomes that promote its activation and protect it against E3 ligase FBXL16-mediated degradation. FBXL16 binds to the PRM region of LATS2, disrupting condensate formation and facilitating LATS2 degradation, the loss of condensate integrity directly contributes to tumor progression [250]. Similarly, the lipid-associated lncRNA SNHG9, together with phosphatidic acid, promotes the condensation of LATS1 to form inactive droplets, thereby attenuating its suppressive effect on YAP and facilitating breast cancer progression [251]. At the downstream effector level, YAP forms co-condensates with the coactivator SRC-1, maintaining an active pool of nuclear signals. This structure can be selectively dismantled by targeted pharmacologic agents, offering a novel strategy for the precise inhibition of YAP oncogenic activity and the development of LLPS-targeted therapies [252]. Collectively, these findings demonstrate that biomolecular phase separation precisely orchestrates the transmission and termination of Hippo pathway signaling.

cGAS–STING pathway

The cGAS–STING signaling pathway acts as a pivotal intracellular defense mechanism against both viral infection and tumorigenesis (Fig. 6b). Upon recognizing cytosolic double-stranded DNA, cGAS leverages its positively charged intrinsically disordered N-terminus to mediate multivalent interactions with DNA, driving the formation of liquid-like condensates. This process markedly enhances the binding affinity of cGAS for DNA [253, 254]. The propensity of cGAS–DNA LLPS can be further potentiated by factors such as long-chain DNA, free zinc ions, specific nucleic acid modifications, as well as bacterial proteins like streptavidin, enabling sensitive immune surveillance even under conditions of low DNA abundance [255,256,257]. Although cytosolic RNA does not directly activate cGAS, it can facilitate the co-phase separation of cGAS with DNA, which is particularly effective at enhancing interferon signaling when dsDNA concentrations are limited [256]. Importantly, while LLPS establishes a spatial platform for cGAS activation, it simultaneously creates vulnerabilities that can be exploited by metabolic byproducts and viral proteins. Tumor-derived metabolites such as acetaldehyde and oleic acid are capable of dissolving cGAS–DNA condensates, dispersing these signaling hubs and attenuating downstream immune responses [258, 259]. Moreover, N-terminal lysine lactylation of cGAS, induced by lactate, disrupts its phase separation with DNA, shifting cGAS into an inactive gel-like state [95]. Some viral proteins, such as KicGAS, can oligomerize and bind DNA to form decoy condensates, thereby efficiently blocking the initial activation of cGAS signaling [260]. It is also notable that deletion of the IDRs from cGAS results in the formation of solid-like aggregates and a complete loss of catalytic activity, further underscoring the essential role of the IDR in functional condensation and immune activation [261]. Downstream, STING also undergoes LLPS to form membrane-like biocondensates at the endoplasmic reticulum, modulating its own activity and that of TBK1. This spatial buffering mechanism is critical for preventing excessive inflammation but may be hijacked by tumor cells to suppress interferon production and evade immune surveillance [262]. At the effector stage, proteins such as AXIN1, TBK1, and USP35 can assemble into LLPS-mediated signalosomes, stabilizing IRF3 and promoting type I interferon expression to enhance antiviral immunity [263]. Conversely, mutations in the NF2 gene can induce the formation of aberrant condensates that recruit PP2A, inactivating TBK1 and weakening antitumor immunity [72]. In summary, biomolecular phase separation orchestrates the cGAS–STING signaling pathway by regulating the assembly, spatial distribution, and functional precision of its key components. This not only ensures efficient and timely innate immune surveillance and activation but also provides novel mechanisms for immune evasion and dysregulation by tumors, viruses, and metabolic disturbances.

p53 pathway

As a classic tumor suppressor, p53 is mutated in more than half of malignant tumors, establishing it as a pivotal molecule in cancer research [264,265,266]. Recent studies have demonstrated that p53 can assemble membrane-less nuclear condensates through phase separation, a process orchestrated by its IDR and tetramerization domain, and facilitated by electrostatic and hydrophobic interactions [267]. In response to DNA damage, wild-type p53 undergoes LLPS to initiate transcription of downstream target genes, thereby mediating stress responses and tumor suppressive functions [76]. However, mutations in the tetramerization domain can hinder tetramer formation, resulting in reduced or abolished LLPS capability, impaired target gene activation, and consequently increased cell survival and tumorigenesis risk [76]. In addition to tetramerization domain mutations, cancer-associated mutations in the DNA-binding domain, such as M237I and R249S, also affect p53 phase separation. These mutations enhance the LLPS propensity of p53 and accelerate liquid-to-solid phase transitions, increasing hydrophobic exposure and weakening intramolecular interactions, which ultimately lead to aberrant signaling [27]. This mechanism is believed to underpin the oncogenic gain-of-function exhibited by p53 aggregates, offering new therapeutic targets for anti-cancer drug development [27]. Notably, impairment of p53 LLPS function is not limited to genetic mutations, the metabolic environment also plays a significant role. Recent studies have identified AARS1 as a lactate sensor that mediates lysine lactylation in tumor cells, including at K120 and K139 within the DNA-binding domain of p53 (Fig. 6c). This lactylation modification disrupts p53 phase separation, DNA binding, and transcriptional activation, leading to compromised tumor suppressor function [94]. Targeting the AARS1–lactate axis, such as by using β-alanine to block lactate binding to AARS1, holds promise for restoring p53 function and inhibiting tumor progression. In summary, biomolecular phase separation represents a novel perspective for understanding p53 dysfunction and tumorigenesis.

Wnt/β-catenin pathway

The Wnt/β-catenin signaling pathway utilizes biomolecular phase separation to regulate the spatial activation and inactivation of signaling events within different cellular compartments (Fig. 6d). At the pathway’s initiation, the assembly of both the membrane-localized signalosome and the cytoplasmic β-catenin destruction complex is critically dependent on condensation. For example, the scaffold protein Dishevelled 2 (Dvl2) undergoes LLPS via its IDR, forming signalosomes that are further organized through interactions with receptors such as Fzd5 [268]. Notably, even in the absence of ligand stimulation, Dvl2-mediated LLPS can enable the dynamic, low-concentration recruitment of Axin1 to the signalosome, attenuating Axin’s phase separation and disrupting the β-catenin destruction complex. This enables β-catenin signaling to be activated independently of classical Wnt ligands [268, 269]. Conversely, the formation of the destruction complex is also orchestrated by condensation. The IDR of Axin promotes its phase separation, allowing the assembly of the destruction complex with key enzymes such as APC, GSK3β, and CK1α, which ensures efficient phosphorylation and degradation of β-catenin [75, 270]. The LLPS capacity of APC is modulated by its 20-amino acid repeat and self-association domains, and can be dynamically regulated by site-specific phosphorylation, acting as a molecular switch for condensate formation [271]. In colorectal cancers, APC mutations do not block condensate formation but impair the recruitment of components such as GSK3β and CK1α, thereby hindering β-catenin phosphorylation and degradation, and resulting in abnormal cytoplasmic accumulation and oncogenic transcriptional activation [272]. Additionally, regulatory factors such as USP10 can promote the physical interaction and phase separation of Axin1 and β-catenin, spatially restricting β-catenin activity. This mechanism inhibits aberrant β-catenin accumulation and pathological amplification of Wnt signaling across various physiological and disease contexts [273].

TGF-β pathway

The accuracy and robustness of TGF-β pathway signaling output also critically depends on the involvement of LLPS. At the level of signaling crosstalk, TGF-β stimulation induces upregulation of DACT1, which utilizes its IDRs to form distinct phase-separated condensates within the cytoplasm. These condensates selectively sequester the Wnt pathway activator CK2, thereby precisely repressing Wnt signaling and maintaining the anti-metastatic effects mediated by TGF-β [274]. Loss of DACT1’s LLPS capacity eliminates this inhibitory effect on Wnt signaling, highlighting the essential role of DACT1 condensates in orchestrating crosstalk between signaling pathways. At the level of nuclear transcriptional regulation, the downstream Smad2/3/4 complex also undergoes LLPS, forming phase-separated transcriptional condensates on chromatin (Fig. 6e). These act as transcriptional hubs, particularly at the TAT promoter, markedly enhancing target gene expression and pro-apoptotic signaling [189]. This spatial compartmentalization not only ensures robust Smad-dependent transcriptional activation, but also directly inhibits tumor cell proliferation and promotes apoptosis, highlighting the role of biomolecular phase separation in reinforcing the tumor-suppressive function of the TGF-β pathway. However, the spatial organization mediated by condensation also reveals mechanisms of signal attenuation. Recent studies have identified the RBP SFPQ, a prion-like factor frequently upregulated in cancer, as a potent suppressor of TGF-β signaling. Through its PLDs, SFPQ enables LLPS to form nuclear condensates that sequester Smad4, thereby excluding it from the Smad complex and chromatin, and substantially dampening the tumor-suppressive activity of TGF-β signaling [275]. When SFPQ is overexpressed or its LLPS activity is aberrantly enhanced, cellular sensitivity to TGF-β-mediated growth inhibition is significantly reduced, ultimately promoting tumorigenesis and progression.

Other signaling pathways

Beyond classical pathways, biomolecular phase separation exhibits unique regulatory mechanisms in multiple oncogenic signaling cascades. In the Ras/MAPK signaling pathway, studies have demonstrated that RET fusion oncoproteins, such as CCDC6-RET, utilize LLPS to form a ternary signal niche with GRB2 and SHC1 (Fig. 6f). This phase-separated compartment, dependent on both the kinase domain of the fusion protein and its partner, significantly promotes RET fusion autophosphorylation and kinase activity. Consequently, the efficiency of the downstream signaling cascade is heightened, providing a mechanistic explanation for the persistent activation of the Ras/MAPK pathway [276]. Similarly, in EML4-ALK–driven lung cancer, different variants of EML4-ALK can assemble LLPS-based cytoplasmic signal complexes centered on MAPK, PLCγ, and PI3K. Studies indicate that the assembly and stability of these condensates rely not only on the ALK kinase domain but can also be modulated in a dynamic manner by different ALK inhibitors. This highlights the structural plasticity and pharmacological sensitivity of BMCs in oncogenic signaling [277]. Furthermore, investigations into the Notch pathway demonstrate that the Notch1 intracellular domain, owing to its IDRs, can undergo nuclear phase separation to form transcriptional condensates. These structures recruit and concentrate transcriptional machinery, coordinate enhancer-promoter looping, and facilitate the expression of the MYC oncogene. These LLPS-driven transcriptional condensates not only enhance the expression efficiency of Notch target genes but also provide structural fidelity for high-precision signal transmission [278]. In addition, in the STAT3 signaling pathway, EZH2 protein, upon acquiring hydrophobicity through N-myristoylation, readily undergoes LLPS to form specific condensates that compartmentalize and activate STAT3, promoting rapid proliferation of tumor cells [279]. Collectively, these findings underscore that biomolecular phase separation constitutes a novel theoretical framework for dissecting oncogenic signal transduction and presents promising opportunities for the development of targeted therapeutic interventions.

Table 3 Tumor therapeutic strategies based on biomolecular phase separation

Full size table

Application of biomolecular phase separation in cancer therapy

In recent years, biomolecular phase separation has emerged as a forefront topic in biomedical drug discovery. Studies investigating the induction of condensation to promote tumor cell death and inhibition of condensation to overcome drug resistance have both demonstrated substantial clinical promise (Table 3). A variety of approaches, including small-molecule modulators, protein engineering, and advanced materials, are accelerating the translation of biomolecular phase separation from fundamental mechanistic studies to targeted therapeutic applications across various diseases. Biomolecular phase separation holds great potential to revolutionize therapies for intractable diseases and is expected to become a promising frontier in drug discovery and translational medicine.

Promote biomolecular phase separation

Promoting biomolecular phase separation can induce the formation or remodeling of functional condensates inside cells. This process enables precise control over signaling pathways, cell death, and metabolism. It offers new therapeutic strategies for challenging diseases such as cancer (Fig. 7a). Inducing phase separation of proteins or nucleic acids has been shown to trigger tumor cell death. For example, in AML, the RBP fibrillarin regulates pre-rRNA processing through its phase separation domain, which is critical for AML cell survival. Targeting fibrillarin with the chemotherapeutic agent CGX-635 induces fibrillarin aggregation, disrupting pre-rRNA processing and significantly reducing AML cell viability [280]. In liver cancer, the small molecule RQ induces β-catenin to form condensates in the cytoplasm, preventing its entry into the nucleus and subsequent activation of oncogenes. This strategy not only effectively suppresses the oncogenic function of β-catenin but also successfully avoids immune evasion, providing a new approach for liver cancer treatment [281]. Additionally, cationic polymers (CPs), as inducers of RNA phase separation, exhibit great potential in cancer therapy. CPs induce RNA phase separation, encapsulating TGFβ1 mRNA in the RNA droplets, thereby reducing the immune suppression of tumor cells. This approach demonstrated marked anti-tumor effects in a mouse breast cancer model [282]. In pancreatic ductal adenocarcinoma, ETS homologous factor (EHF) could form liquid-like condensates via its phase separation properties, inhibiting the transcription of telomerase reverse transcriptase, thereby shortening telomere and inducing cellular senescence. Preclinical studies revealed that Bilobetin, an EHF phase separation enhancer, not only promotes EHF phase separation but also ameliorates the immunosuppressive tumor microenvironment, sensitizing pancreatic ductal adenocarcinoma to anti-PD-1 therapy [283]. In breast cancer, the small molecule XS561 serves as a selective Nur77 ligand. This binding promotes Nur77 translocation from the nucleus to the mitochondria, where Nur77 forms condensates with BCL-2, triggering mitochondria-mediated apoptosis [284]. In multiple myeloma, all-trans retinoic acid (ATRA) enhances the phase separation properties of RARα by binding to it. As a result, RARα interacts more with NSD2, leading to an increase in CD38 gene expression and improving the efficacy of anti-CD38 CAR-T cell therapy [285]. Furthermore, the small molecule icFSP1 alters the membrane localization of FSP1, triggering the formation of droplet-like FSP1 condensates through LLPS. This inhibits the function of FSP1 and promotes ferroptosis in tumor cells [286]. Collectively, drugs that induce biomolecular phase separation to form condensates can inhibit the function or alter the survival state of tumor cells, offering a promising and innovative direction for drug discovery and therapeutic development.

Inhibit biomolecular phase separation

LLPS is a frequent driver of pathogenic protein condensate formation. This process can promote transcriptional activation, persistent activation of signaling pathways, or the development of drug resistance. Therefore, precisely inhibiting biomolecular phase separation and its products has become an important strategy for innovative drug development and disease treatment. First, biomolecular phase separation is essential for the pathogenic activities of many signaling molecules, transcription factors, and repair proteins. For example, BRD4, a member of the BET protein family, forms condensates at SE regions through LLPS, which promotes the transcription of oncogenes. The natural compound PCG can directly bind to BRD4 and inhibit its phase separation capability, converting dynamic BRD4 condensates into static aggregates. This process effectively shuts down BRD4-dependent transcriptional programs, offering a novel mechanism for tumor therapy [287]. Similarly, the transcription factor SP1 can assemble SEs through LLPS in lung adenocarcinoma, driving the high expression of oncogenes such as RGS20. The phase separation capability of SP1 can be blocked by the demethylase inhibitor GSK-J4, which consequently attenuates the SP1-driven tumor phenotype [288]. The transcriptional complex formed by YAP and TEAD can undergo LLPS at SE regions, promoting the high expression of oncogenes. The small-molecule inhibitor ETS-006 effectively disrupts YAP/TEAD-mediated LLPS and downstream oncogenic signaling pathways. ETS-006 has received clinical trial approval in both China and the United States, as well as FDA orphan drug designation, offering new hope for the treatment of various refractory cancers [289]. Inhibition biomolecular phase separation has also emerged as an important strategy to overcome tumor drug resistance. In castration-resistant prostate cancer, the androgen receptor (AR) forms transcriptionally active condensates via LLPS, driving downstream gene expression and sustaining signaling activity even in the presence of antiandrogen therapy, which promotes the development of antiandrogen resistance. The small molecule ET516 can specifically disrupt AR LLPS condensates and effectively reverse drug resistance [290]. In hepatocellular carcinoma, RIOK1 assembles SGs via LLPS with associated proteins, suppressing PTEN expression and facilitating both TKI resistance and tumor progression. Chidamide can downregulate RIOK1 and prevent SG formation, thereby enhancing the therapeutic efficacy of TKIs [150]. Protein–nucleic acid LLPS also represents a novel disease target. Recent studies have demonstrated that arsenic trioxide (ATO) can directly disrupt the condensation between N-Myc and DNA, inhibiting N-Myc transcriptional activity [291, 292]. Moreover, a recent clinical trial revealed that conventional chemotherapy regimens containing ATO may prolong disease stability and exhibit promising efficacy and safety in patients with relapsed or refractory high-risk neuroblastoma (NCT03503864). Additionally, in tumor cells, poly (ADP-ribose) (PAR) synthesized by PARP1 can recruit FUS protein via condensation to form DNA damage repair foci. After DNA repair is complete, PARG degrades PAR, promoting the dissolution of FUS condensates (Fig. 7b) [111]. PARP inhibitors, such as olaparib, reduce the synthesis of PAR chains and block the LLPS process of proteins like FUS, inhibiting DNA repair and increasing the sensitivity of tumor cells to DNA damage. In the immune field, cGAS initiates innate immune responses by undergoing phase separation with DNA. Novel protein condensate inhibitors, such as XL-3156 and XL-3158, can simultaneously bind to both the allosteric and orthosteric sites of cGAS, maintaining it in an inactive state and blocking the formation of cGAS–DNA condensates [293]. Furthermore, protein degradation technologies such as PROTACs provide additional tools for regulating LLPS. For example, BRD4-targeting PROTAC molecules can degrade BRD4, eliminate its SE condensates, suppress oncogene expression, and offer new strategies for the treatment of various cancers [294]. Moreover, NUPR1 can form SGs via condensation, promoting survival and progression of pancreatic cancer cells. The small molecule inhibitor ZZW-115 can inhibit NUPR1 activity, effectively inducing death in Kras-mutant pancreatic cancer cells and providing a new therapeutic avenue for pancreatic cancer [295]. Recent studies have also found that nanomaterials with different chiralities can regulate the assembly of SGs via condensation in tumor cells, thereby enhancing the sensitivity of tumor cells to chemotherapeutic drugs, at the same time, such chiral regulation offers a certain degree of protection to normal cells [296]. In summary, inhibition of biomolecular-phase-separation -related processes through small molecule inhibitors, protein degradation technologies, natural products, and other approaches is emerging as a novel strategy in cancer therapy, and is expected to bring new hope for the treatment of refractory diseases.

Conclusion and challenges

The emergence of biomolecular phase separation theory has revolutionized conventional paradigms of cellular structure organization. Unlike traditional membrane-bound compartments, biomolecular phase separation enables the formation of dynamic BMCs that provide spatiotemporal control over key cellular processes. In cancer research, biomolecular phase separation is now seen as a critical regulatory mechanism, intersecting with multiple pathways and phenotypes linked to tumor development and progression. This review presents a comprehensive overview of the biophysical principles underlying biomolecular phase separation, the molecular factors that govern its formation, and the environmental signals that influence its dynamics. Biomolecular phase separation critically regulates multiple oncogenic processes that mirror the hallmarks of cancer, encompassing uncontrolled proliferation, metastasis, immune evasion and metabolic reprogramming, among others. Importantly, condensation provides a distinct molecular framework for the functional compartmentalization of transcriptional coactivators, SEs, non-coding RNAs, and chromatin-associated factors, many of which have been considered undruggable due to their lack of enzymatic activity or stable structural domains.

Although condensation has been recognized as a key biophysical mechanism in tumorigenesis and cancer progression, several fundamental questions about its mechanisms, methodological challenges, and translational potential remain unresolved. One key issue is the functional distinction between BMCs formed through condensation and traditional protein complexes. It is still unclear whether biomolecular phase separation acts solely as a spatial organizing tool or provides unique, non-redundant regulatory functions in cellular processes. In addition, the ways in which different types of condensates coordinate or interact, both in vitro and in vivo, especially within the complex and dynamic tumor microenvironment, are not yet well understood. Many in vitro studies rely on conditions such as supraphysiological protein concentrations, non-physiological pH, or altered ionic strength, which differ significantly from the intracellular environment and may limit their relevance to clinical settings. Developing physiologically relevant models, including organoid systems and ex vivo tumor slice cultures, is therefore essential to better capture biomolecular phase separation behavior under disease-relevant conditions.

Current technologies for in situ and real-time analysis of endogenous condensate dynamics still face significant limitations. While conventional fluorescence microscopy provides valuable structural information, it often lacks the temporal resolution and sensitivity to fully capture the dynamic spatiotemporal behavior of condensate nucleation, maturation, fusion, and dissolution. Although biophysical methods such as fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and atomic force microscopy can probe specific aspects of condensate properties, their application in live animal models or complex tumor microenvironments remains technically challenging due to limitations in spatial/temporal resolution and penetration depth. There is a critical need to develop robust imaging platforms that combine high spatial resolution with both minimal invasiveness and precise temporal control. Furthermore, elucidating the internal architecture and molecular composition of condensates in living systems continues to pose significant technical challenges. The synergistic integration of optogenetic manipulation, super-resolution microscopy, and spatially resolved proteomic approaches may provide transformative solutions to these current limitations.

From a disease-focused perspective, it remains unclear whether biomolecular phase separation dysregulation exhibits cell type specific features. Variations in cell lineage and differentiation state may lead to condensates with distinct molecular compositions and regulatory functions, potentially shaping their involvement in oncogenic transformation, progression, or therapeutic resistance. Identifying pathological condensates linked to key cancer traits such as unchecked proliferation, invasion, immune escape, or drug tolerance may clarify whether biomolecular phase separation functions as a true driver of tumorigenesis or reflects a secondary response to cellular stress. This distinction carries important implications for clinical translation. Although a few small molecules such as 1,6-HD and lipoamide have been shown to modulate condensation, their use is limited by poor specificity, off-target effects, and unclear mechanisms of action. As a result, the rational design of therapeutics that directly target BMCs remains a significant unmet need. Future strategies may include the development of ligands that bind IDRs, inhibitors of phase-separation-related PTMs such as phosphorylation and ubiquitination, or modulators that respond to the tumor microenvironment to selectively influence biomolecular phase separation behavior in cancer cells.

The question of whether deliberate targeting of biomolecular phase separation leads to greater clinical translational success than conventional strategies remains controversial. Interestingly, many key proteins and signaling pathways involved in condensation regulation, such as BRD4 and YAP/TAZ, have already been extensively studied as therapeutic targets in traditional drug development [297,298,299,300,301]. This implies that we may have been modulating phase separation mechanisms unintentionally through existing therapeutic approaches. In this light, the growing interest in condensation may serve less as a paradigm shift in drug design, and more as a new mechanistic framework for interpreting established therapeutic strategies. This, in turn, raises a compelling question: have we, perhaps unknowingly, already influenced biomolecular phase separation in early-stage drug screening and mechanistic studies? A deeper investigation into this potential link may reveal previously unrecognized functional dimensions of many classic drug targets.

Beyond mechanistic insights, it is worth emphasizing that biomolecular phase separation holds potential for applications other than simply targeting intracellular condensates. Increasing evidence supports the emerging role of phase separation in drug delivery and the design of nanoscale biomaterials. Recent studies have highlighted notable advances in LLPS-based delivery systems. For instance, researchers have developed a drug–DNA–histone complex that spontaneously forms condensates within tumor cells, enabling sustained drug release and efficient intratumoral accumulation, thereby significantly enhancing the therapeutic efficacy against drug-resistant cancers [302]. In another study, cholesterol-modified DNA and histones cooperatively induced LLPS, resulting in vesicle-like structures that function without lipid membranes and efficiently deliver mRNA, viruses, and cytokines. In animal models, this platform not only promoted tumor cell oncolysis but also robustly activated antitumor immune responses [303]. These findings suggest that biomolecular phase separation represents not only a valuable framework for understanding cancer biology but also a promising foundation for the development of functional biomaterials and precision delivery technologies in future cancer therapies.

Biomolecular phase separation research is inherently interdisciplinary and demands collaboration across molecular biology, structural biology, biophysics, systems biology, and computational modeling. Establishing standardized experimental criteria, expanding curated databases of biomolecular phase separation related proteins, and advancing high-throughput screening platforms for disease-relevant condensates will be essential for translating condensation from a biophysical concept into a clinically actionable framework in oncology.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

AD:: Activation domain
ALS:: Amyotrophic lateral sclerosis
ALT:: Alternative lengthening of telomeres
AML:: Acute myeloid leukemia
APA:: Alternative polyadenylation
APB:: ALT-associated promyelocytic leukemia body
AR:: Androgen receptor
ATO:: TOA
ATP:: Adenosine triphosphate
ATRA:: All-trans retinoic acid
BMC:: Biomolecular condensate
CAR:: Chimeric antigen receptor
CC:: Coiled-coil
CPs:: Cationic polymers
DMN-SIPL:: Hepsin-recognized amphiphilic-branched peptide
DSB:: Double-strand breaks
Dvl2:: Dishevelled 2
ECs:: Endothelial cells
EHF:: ETS homologous factor
ELP:: Elastin-like polypeptide
EMT:: Epithelial-mesenchymal transition
FSP1:: Suppressor protein 1
HK:: Hexokinase
IDRs:: Intrinsically disordered regions
LC:: Low-complexity
LLPS:: Liquid-liquid phase separation
lncRNA:: Long non-coding RNA
m6A:: N6-methyladenosine
MOVLD:: Multi-organ venous and lymphatic defect
PAR:: Poly ADP-ribose
PLD:: Prion-like domain
Pol II:: Polymerase II
PTM:: Post-translational modification
RMDs:: Modular domains
RBP:: RNA-binding proteins
SE:: Super-enhancer
SG:: Stress granule
1,6-HD:: 1,6-hexanediol

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Acknowledgements

The figures in this article were supported by BioRender.

Funding

This work was supported by the Guangdong Basic and Applied Basic Research Foundation (Grant No. 2024A1515012793), Guangdong Provincial Medical Science Research Fund Project (Grant No. B2025107, No. B2025114), and the National Natural Science Foundation of China (Grant No. 82273404).

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Lan Hu and Zikun Huang contributed equally to this work.

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Department of Radiotherapy, Cancer Hospital of Shantou University Medical College, No. 7 Raoping Road, Shantou, Guangdong, 515041, China
Lan Hu & Ying Zhang
Department of Orthopedics, the First Affiliated Hospital of Shantou University Medical College, No.57 Changping Road, Shantou, Guangdong, 515031, China
Zikun Huang & Zhaoyong Liu

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Lan Hu
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Zikun Huang
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Zhaoyong Liu
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Ying Zhang
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L.H. drafted and conceived the initial manuscript. ZK.H. drew the figures and arranged the tables. Y.Z. and ZY.L. revised the manuscript. All authors have read and approved the article.

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Correspondence to Zhaoyong Liu or Ying Zhang.

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Hu, L., Huang, Z., Liu, Z. et al. Biomolecular phase separation in tumorigenesis: from aberrant condensates to therapeutic vulnerabilities. Mol Cancer 24, 220 (2025). https://doi.org/10.1186/s12943-025-02428-1

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Received: 18 June 2025
Accepted: 31 July 2025
Published: 23 August 2025
Version of record: 23 August 2025
DOI: https://doi.org/10.1186/s12943-025-02428-1

Biomolecular phase separation in tumorigenesis: from aberrant condensates to therapeutic vulnerabilities

Abstract

Introduction

The history of biomolecular phase separation

Structural basis of biomolecular phase separation

IDR

Multiple folded domains

Regulatory factors of biomolecular phase separation

Intrinsic factors

Protein concentration

Protein structure

Gene mutations

Gene fusion

PTMs

Multicomponent interactions

Extrinsic factors

Temperature

pH

Salt

ATP

Stress

Metabolites

Molecular chaperones

Nucleic acids

Biomolecular phase separation regulates the hallmarks of cancer

Biomolecular phase separation regulates tumor proliferation

Biomolecular phase separation regulates tumor metastasis

Biomolecular phase separation regulates tumor cell death

Biomolecular phase separation regulates the evasion of growth suppressors in tumor cells

Biomolecular phase separation regulates tumor angiogenesis

Biomolecular phase separation supports the unlimited replicative potential of tumor cells

Biomolecular phase separation regulates tumor cell phenotypic plasticity

Biomolecular phase separation regulates tumor energy metabolism

Biomolecular phase separation regulates tumor immune destruction

Biomolecular phase separation regulates tumor epigenetic reprogramming

Molecular mechanisms of biomolecular phase separation

Gene transcription regulation

Maintain genome stability

Regulation of signal transduction

Hippo pathway

cGAS–STING pathway

p53 pathway

Wnt/β-catenin pathway

TGF-β pathway

Other signaling pathways

Application of biomolecular phase separation in cancer therapy

Promote biomolecular phase separation

Inhibit biomolecular phase separation

Conclusion and challenges

Data availability

Abbreviations

References

Acknowledgements

Funding

Author information

Authors and Affiliations

Contributions

Corresponding authors

Ethics declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Additional information

Publisher’s Note

Rights and permissions

About this article

Cite this article

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Keywords

Molecular Cancer

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