Abstract Actin-rich protrusions densely cover the surface of T cells and are well characterised for their role in migration. Recent studies have uncovered their contribution to antigen surveillance and immune signalling. To further explore how protrusions initiate signalling pathways mediating T-cell activation, we performed live-cell imaging of endogenously tagged proteins in HER2-specific chimeric antigen receptor (CAR) T cells targeting HER2⁺ breast-cancer cells. Quantitative STED microscopy allowed us to monitor protein rearrangement and to correlate it with membrane topology over time. Before activation, key signalling proteins (including Lck, CD45, LAT, and the CAR) were not enriched in protrusions. Upon contact with target cells, rapid protein reorganisation occurred preferentially within protrusions, initiating signalling. HER2-CAR clustering, accompanied by ZAP-70 and LAT recruitment, was enhanced in protrusions. While Lck distribution remained unchanged, exclusion of the phosphatase CD45 was enhanced at protrusion-cell contacts, independently of the CAR signalling domain. Overall, signalling machinery rearranged faster and more effectively at protrusive contacts than at main plasma membrane regions. Together, our data re-frame protrusions as sites of enhanced receptor activation by exclusion and clustering dynamics rather than by pre-enrichment of the signalling machinery.

Abstract Background and Significance: Diffuse large B-cell lymphoma, activated-B-cell-like type (ABC-DLBCL) is marked by chronic BCR and NF-κB signaling, which are often the result of activating mutations of CD79B, MYD88 and CARD11. In addition, deleterious mutations in negative regulators of BCR signaling, including PKCδ, SHP1 and SHIP, are found in 5-10% of DLBCL. PKCδ and the phosphatases SHP1 and SHIP1 curb excessive B-cell activation and defects in these molecules result in autoimmunity. Results: Here, we discovered a mechanistic framework of dynamic BCR-feedback control in activated and germinal center (GC) B-cells that is frequently inactivated in DLBCL. This feedback control depends on coordinated activity of PKCδ, the inhibitory phosphatases SHP1 and SHIP1, and the surface receptor CD25 in assembling inhibitory complexes in proximity of BCR. Proteomic analysis revealed strong induction of PKCδ and CD25 expression upon BCR engagement. Genetic engineering of murine and human B cells identified PKCδ and CD25 as negative regulators of BCR and NF-κB signaling. RNA-seq and phosphoproteomic studies revealed that ablation of PKCδ or CD25 in B cells resulted in prominent activation of NF-kB gene expression programs and hyperphosphorylation of multiple substrates in the BCR-signaling pathway. Conversely, both PKCδ- and CD25-ablation caused loss of phosphorylation of inhibitory phosphatases SHP1 and SHIP1. Mechanistic studies showed that oncogenic BCR signaling induced PKCd-mediated CD25-phosphorylation on its cytoplasmic tail (S268, T271). To understand the functional importance of PKCδ-mediated CD25 phosphorylation, we introduced genetic CD25 knockin alleles using HDR templates encoding wildtype CD25 or CD25-S268A/T271V (AV) mutations in human GC B-cells. Interestingly, CD25-AV knockin GC B-cells failed to terminate BCR-signaling and showed autonomous Ca2+ oscillations. In addition, CD25-AV knockin GC B-cells expressed increased levels of activation markers CD69, CD80 and CD86, and exhibited constitutive activation of the NF-kB pathway. In coculture experiments determining how CD25 regulates competitive fitness, CD25-AV knockin GC B-cells rapidly outcompeted their CD25-wildtype counterparts. Interactome studies and molecular dynamics simulations revealed that PKCδ-dependent phosphorylation of CD25 was required to retain inhibitory phosphatases in proximity of BCR signaling complexes to attenuate BCR signaling. Moreover, defective PKCδ-induced assembly of inhibitory complexes was restored with a newly developed bispecific antibody and chemically induced heterodimerization that directed SHP1 towards the BCR. Conclusion: DLBCLs frequently harbor activating mutations that sustain oncogenic BCR signaling as well as deleterious mutations of inhibitory phosphatases. Our findings show that PKCδ-mediated CD25 phosphorylation is required for assembly of inhibitory complexes in the proximity of BCR, which mediates constitutive BCR signaling suppression in GC B cells but is inactivated in DLBCL to enable unconstrained signaling activity and proliferative advantage. Citation Format: Ruifeng Sun, Jaewoong Lee, Kathrin Kläsener, Mark E. Robinson, Kohei Kume, Zhangliang Cheng, Kadriye N. Cosgun, Etienne Leveille, Longhui Zeng, Xiaolei Su, Ning Ma, Nagarajan Vaidehi, Huimin Geng, Eric Meffre, Daniel J. Hodson, Mathhew S. Davids, Michael Reth, Markus Müschen. PKCδ-mediated feedback control of B-cell receptor signaling and its subversion in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Fifth AACR International Meeting on Advances in Malignant Lymphoma: From Discovery to Clinical Impact; 2026 Jun 24-27; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2026;7(3_Suppl):Abstract nr A009.

Plasma membrane-associated condensates driven by liquid-liquid phase separation represent a novel mechanism of receptor-mediated signaling transduction, serving as mesoscale platforms that concentrate signaling molecules and modulate reaction kinetics. Condensate formation is a highly dynamic process that occurs within seconds to minutes following receptor activation. Here, we present methods for de novo reconstituting liquid-like condensates on supported lipid bilayers and assessing the condensate fluidity using fluorescence recovery after photobleaching (FRAP). This protocol encompasses supported lipid bilayer preparation, condensation imaging, and FRAP analysis using total internal reflection fluorescence (TIRF) microscopy. Supported lipid bilayers provide a membrane-mimicking environment for receptor signaling cascades, offering mechanistic insights into protein-protein and lipid-protein interactions amid micron-scale condensates. The protocol can also be adapted to study condensates associated with the internal membranes of the Golgi apparatus, mitochondria, and other organelles. Key features • Real-time imaging of condensate formation and FRAP analysis using TIRF microscopy reveals a spatiotemporal profile of signaling transduction. • Supported lipid bilayers provide a fluidic membrane environment that is critical for the biochemical reconstitution of condensates at physiological protein concentrations. • The lipid and protein components within the reconstituted condensate system can be readily manipulated to accommodate specific experimental objectives and assay designs.

Abstract Introduction Biomolecular condensation provides a fundamental mechanism for organizing signaling complexes within cells. However, its occurrence on the cell surface–rich in glycans and exposed to dynamic extracellular forces–remains poorly understood. Fractalkine (CX3CL1), an endothelial membrane-bound chemokine, is heavily O-glycosylated and interacts with the monocyte receptor CX3CR1. Although fractalkine is known to mediate monocyte adhesion and migration, how its glycosylation drives condensate formation and endothelial signaling has not yet been elucidated. Methods We combined live-cell imaging, biochemical reconstitution, and a newly applied mucinase-based O-glycoproteomic approach to dissect the mechanism of fractalkine condensate formation. This strategy enabled site-specific mapping of O-glycosylation within the mucin domain and identification of key sialylated residues critical for condensate assembly. Functional analyses using glycosylation-deficient HUVEC mutants, including transendothelial migration assays, were performed to evaluate the impact of glycans on condensate formation and endothelial signaling. Results We found that endothelial membrane-bound fractalkine forms glycan-dependent condensates at the monocyte—endothelium interface. CX3CR1 engagement induces these condensates in a glycan-dependent manner. Glycoproteomic mapping revealed that a specific site of sialylated O-glycans is critical for these processes. The formation of these endothelial fractalkine condensates promotes the recruitment and activation of VEGFR2, leading to calcium influx and the upregulation of adhesion molecules, thereby enhancing monocyte transendothelial migration. Conclusion Our findings uncover a sialylation-dependent biomolecular condensate that mediates bidirectional fractalkine—CX3CR1 signaling. By recruiting and activating VEGFR2, this condensate links cell-surface glycosylation to endothelial signaling and immune cell migration, revealing a new paradigm of glycan-controlled cell-surface condensation. Funding Source n/a Topic Categories Cytokines and Chemokines and their Receptors (CCR)

Biomolecular condensates are membraneless bodies that organize biochemical reactions typically within cells. However, the roles of condensates in extracellular space-where conditions differ substantially from intracellular space-remain poorly understood. Here we report that mast cell extracellular granules (MCEGs), a stable membraneless entity, are condensates assembled through electrostatic interactions between glycosaminoglycans and polyamines. Disrupting polyamine synthesis or trafficking blocks MCEG formation and compromises the storage of proteases and cytokines. Granules reconstituted with heparin and spermine are sufficient to enrich mediators such as carboxypeptidase A3 (CPA3) and tumor necrosis factor (TNF), maintaining an elevated pH and higher concentrations of calcium and zinc compared to the extracellular milieu. This unique environment enhances CPA3 enzymatic activity. Furthermore, the granules increase TNF binding and its bioactivity toward endothelial cells. Together, we reveal MCEGs as functionally active biomolecular condensates with distinct biochemical and immunological properties; MCEGs are formed through sugar-metabolite interactions, expanding the mechanisms of condensate assembly beyond classical protein-protein and protein-RNA interactions.

Chimeric antigen receptor (CAR)-T cell therapies have shown remarkable efficacies for treating otherwise intractable cancers. However, current clinically approved CAR-T therapies are limited by low antigen sensitivity, impeding their efficacy against cancers with low antigen expression. Here, to address this issue, we engineered CARs targeting CD19, CD22 and HER2 by including intrinsically disordered regions (IDRs) that promote signaling condensation. We discovered that the CAR fused with an IDR from FUS, EWS or TAF15 promoted the formation of CAR-T conjugation with cancer targets, the mechanical strength of CAR-T synapses and membrane-proximal signaling, which led to an increased release of cytotoxic factors and a higher killing activity toward low-antigen-expressing cancer cells in vitro. Moreover, the FUS IDR CAR-T induced improved antitumor effects in both blood cancer and solid tumor models. No spontaneous activation in the absence of antigen was observed. Together, our work demonstrates IDRs as a new toolset for improving CAR-T function through inducing biomolecular condensation.

The subcellular localization of mRNAs plays a pivotal role in biological processes, including cell migration. For instance, b-actin mRNA and its associated RNA-binding protein (RBP), ZBP1/IGF2BP1, are recruited to focal adhesions (FAs) to support localized b-actin synthesis, crucial for cell migration. However, whether other mRNAs and RBPs also localize at FAs remains unclear. Here, we identify hundreds of mRNAs that are enriched at FAs (FA-mRNAs). FA-mRNAs share characteristics with stress granule (SG) mRNAs and are found in ribonucleoprotein (RNP) complexes with the SG RBP. Mechanistically, G3BP1 binds to FA proteins in an RNA-dependent manner, and its RNA-binding and dimerization domains, essential for G3BP1 to form RNPs in SG, are required for FA localization and cell migration. We find that G3BP1 RNPs promote cell speed by enhancing FA protein mobility and FA size. These findings suggest a previously unappreciated role for G3BP1 RNPs in regulating FA function under non-stress conditions.

The adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is effective in B cell malignancies. However, the persistence of cancer cells with low levels or complete absence of the target antigen, thereby evading detection by CAR T cells, leads to relapse. These evasion mechanisms highlight the need for receptors with enhanced sensitivity and multispecificity. We introduce a synthetic chimeric T cell receptor (ChTCR) that confers superior antigen sensitivity compared with CARS and previous hybrid TCR designs and is readily adapted for bispecific targeting. ChTCRs replicate the structure of natural TCRs, form classical immune synapses and demonstrate TCR-like signaling. T cells expressing bispecific ChTCRs (Bi-ChTCRs) are more effective than bispecific CAR T cells in eradicating tumors with heterogeneous antigen expression in vivo in female mice. The Bi-ChTCR architecture is resilient and can be designed to target pairs of B cell and multiple myeloma antigens. These findings provide a widely applicable strategy to combat tumor heterogeneity and prevent relapse. Simon, Bugos and colleagues report the design and characterization of monospecific and bispecific synthetic chimeric T cell receptors, conferring increased antigen sensitivity and antitumor activity in B cell malignancies and multiple myeloma models.

Biomolecular condensation has emerged as a general principle in organizing biological processes, including immune response. Xu and colleagues recently reported that the cytoplasmic tail of the CD3ε subunit of the T-cell receptor complex, when fused to a chimeric antigen receptor (CAR), can promote CAR condensation by liquid-liquid phase separation. Through sequence engineering, the authors identified modified CD3ε sequences that enhance the maturation of the immunologic synapse and coreceptor signaling, leading to an improvement in cytotoxicity in vitro and antitumor effects in mouse xenograft models. These results demonstrated that biomolecular condensation could be exploited to improve the function of CAR-T cells, highlighting an exciting strategy for developing next-generation cell therapies.

Phospholipase C gamma 1 ( PLCG1 ) has been identified as the most frequently mutated gene in adult T-cell leukemia/lymphoma, suggesting a critical function of PLCG1 in driving T cell activation. However, it remains unclear how these mutations regulate T cell physiology and pathology. Here, we investigate three common leukemia/lymphoma-associated mutations (R48W, S345F, and D1165H). We discover that these mutations induce hyperactive T cell signaling and cause pro-survival phenotypes. PLCG1 mutants enhance LAT condensation, calcium influx, and ERK activation. They also promote T cell proliferation, upregulate cell adhesion molecules, induce cell aggregation, and confer resistance to Vorinostat, an FDA-approved drug for cutaneous T-cell lymphoma. The resistance depends on ERK signaling and can be reversed with an ERK inhibitor. Interestingly, PLCG1 mutants also induce bystander drug resistance in nearby cells expressing wild-type PLCG1. Mechanistically, alpha smooth muscle actin, which is specifically induced by PLCG1 mutants, directly binds PLCG1 to promote its activation. These results demonstrate that hyperactive PLCG1 promotes T cell survival and drug resistance by inducing non-canonical signaling.

Chimeric antigen receptor (CAR) T cells have demonstrated unprecedented success in treating relapsed or refractory blood cancers. Previous studies of the mechanisms underlying the interactions and responses of CAR T cells and their targets have largely ignored the responses of tumors to CAR ligation. We compared the signaling of a second-generation, ligand-based CAR built from colony-stimulating factor 1 (CSF1) to target the CSF1 receptor (CSF1R) on target cells with a conventional, single-chain variable fragment-based CAR against the B cell antigen CD19. Using SILAC coculture with phosphotyrosine enrichment and LC-MS/MS analysis, we showed that ligation of CSF1R-expressing THP-1 cells with CSF1R-CAR T cells stimulated CSF1R-like signaling in the THP-1 cells. In contrast, no target cell signaling response was observed after the ligation of CD19-CAR T cells with target Raji cells. Using small-molecule inhibitors of the tyrosine kinase Lck, actin polymerization, and CSF1R, we found that CAR-induced CSF1R signaling in THP-1 cells depended exclusively on the kinase activity of CSF1R with no participation from T cell activation. Consistently, CSF1R-CAR T cells promoted THP-1 cell proliferation at low effector-to-target ratios but prevented THP-1 cell proliferation at high effector-to-target ratios. Our data provide evidence for CAR-induced signaling in target cells, an unintended consequence of CARs that may have implications for the choice of CAR antigen for optimal clinical efficacy.

Although chimeric antigen receptor (CAR) T cell therapy has revolutionised individualised cancer therapies for relapsed/refractory lymphomas, low long-term retention due to basal signalling (antigen-independent activation in the absence of cognate antigen) and off-target toxicity limit the broad applicability of CAR-T products. During CAR development, researchers use model systems, like Jurkat T cells (Jurkats), to screen intracellular signalling arrangements based on their ability to activate (e.g., CD69 expression) and withstand repeated antigen encounters. Although Jurkats are standard for CAR screening, the mapping of CAR generations to Jurkat-specific pTyr networks relative to key TCR nodes and CD69 readouts is not well defined, blurring how hierarchical signalling drives activation. Here, we investigated how costimulation influenced tyrosine phosphorylation cascades using LC-MS/MS based phosphotyrosine (pY) proteomics and CD69 expression in the presence of small molecule inhibitors of key TCR signalling regulators. We found that including TCRζ (CD3ζ; gene CD247) in first (ζ-CAR), second (28ζ-CAR and BBζ-CAR), and third (28BBζ-CAR) generation CARs largely determined pY signalling, irrespective of costimulation. Further, we showed that the phosphatase activity of PTPN22 and SHP-1 were largely negligible for activation of CARs, but indiscriminate inhibition of phosphatases using pervanadate (PV) selectively activated BBζ-CARs without antigen encounter. Finally, we found that selective, partial inhibition of Itk using Soquelitinib reduced basal CD69 expression in CAR-Jurkat cells while maintaining their ability to activate in response to antigen. These data suggest that TCRζ determines the pY signalling profile and that Itk drives basal activation of CD19-CAR Jurkats, which may impact evaluation of new CAR designs in CAR-Jurkat screens.

Intrinsically disordered regions (IDRs) of proteins are defined by molecular grammars. This refers to IDR-specific non-random amino acid compositions and non-random patterning of distinct pairs of amino acid types. Here, we introduce grammars inferred using NARDINI+ (GIN) as a resource that uncovers IDR-specific and IDRome-spanning grammars. Using GIN-enabled analyses, we find that specific IDR features and GIN clusters are associated with distinct biological processes, intra-cellular localization preferences, specialized molecular functions, and functionalization as assessed by cellular fitness correlations. IDRs with exceptional grammars, defined as sequences with high-scoring non-random features, are harbored in proteins and complexes that enable spatial and temporal sorting of biochemical activities within the nucleus. Overall, GIN can be used to extract sequence-function relationships of individual IDRs or clusters of IDRs, to redesign extant IDRs or design de novo IDRs, to perform evolutionary analyses through the lens of molecular grammars and GIN clusters, and to make sense of IDR-specific disease-associated mutations.

Actin-rich protrusions densely cover the surface of T cells and are well characterised for their role in cell migration. However, recent studies have uncovered their role in antigen surveillance and immune signalling initiation. To investigate how membrane protrusions initiate and contribute to signalling, from the first cell-cell contact to immunological synapse formation, we performed dynamic imaging experiments of endogenously tagged signalling proteins in T cells. To quantitatively capture the early dynamics of cell-cell interactions, we employed HER2-CAR-expressing T cells targeting HER2 + breast cancer cells. By harnessing live-cell imaging and super-resolution stimulated emission depletion (STED) microscopy we were able to capture topological membrane changes and their correlation with mesoscale protein rearrangements over time. Our findings indicate that, prior to activation, key molecular players in T cell activation, including the kinase Lck, the phosphatase CD45 and the adaptor LAT, as well as the exogenously expressed CAR, lack any enrichment in actin-rich protrusions. However, upon initial contact of the T cell with the target cell, a dynamic and fast rearrangement of the surface receptors, phosphatases, and kinases occurs within the protrusions, ensuring a rapid and effective initiation of the immune signalling cascade. The rapid clustering of the HER2-CAR occurs preferentially within protrusions rather than flat membrane regions and is accompanied by enhanced recruitment of the kinase ZAP-70 and LAT. While the localisation of the kinase Lck remained unchanged, protrusion-cell contacts trigger a pronounced exclusion of the phosphatase CD45, an effect observed both with and without the cytosolic signalling domain of the CAR. Overall, the signalling machinery rearranged more rapidly and efficiently at contacts mediated by protrusive structures compared to non-protrusive regions. Together, our data provide a quantitative framework illustrating how signalling proteins are dynamically reorganised to facilitate CAR-mediated activation within these specialised structures.

Although chimeric antigen receptor (CAR) T cell therapy has revolutionised individualised cancer therapies for relapsed/refractory lymphomas, signalling mechanisms underlying CAR T activation remain incompletely understood, especially among the three generations of CAR T exploiting different signalling domains. Here, using Jurkat T cell as a model, we investigate how costimulation influences tyrosine phosphorylation cascades using LC-MS/MS based phosphotyrosine (pY) proteomics and CD69 expression in the presence of small molecule inhibitors of key TCR signalling regulators. We find that including the ζ-chain in first (ζ-CAR), second (28ζ-CAR and BBζ-CAR), and third (28BBζ-CAR) generation CARs largely determines pY signalling, irrespective of costimulation. Further, we show that the phosphatase activity of PTPN22 and SHP-1 are largely negligible for activation of CARs, but indiscriminate inhibition of phosphatases using Pervanadate (PV) selectively activates BBζ-CARs without antigen encounter. Finally, we find that selective, partial inhibition of Itk using Soquelitinib reduces basal CD69 expression in Jurkat CAR T cells while maintaining their ability to activate in response to antigen. Our data suggest that the ζ-chain determines the pY signalling profile of CD19-CAR Jurkat T cells and that Itk may drive antigen-independent CD19-CAR activation.

Abstract Chimeric antigen receptor (CAR)-T cell-based therapies demonstrate remarkable efficacy for the treatment of otherwise intractable cancers, particularly B-cell malignancies. However, CAR-T cells respond to only a subset of cancers and are limited by low signaling efficiency: a few hundred or thousand antigen molecules are required to activate a CAR T cell whereas a single peptide-loaded MHC molecule is sufficient to trigger the activation of a normal T cell. This low antigen sensitivity not only limits the current CAR-T therapy to high antigen-expressing cancers, but also results in high frequencies of relapse, during which high antigen-expressing cancers downregulate their antigen levels to escape CAR-T’s attacking. To improve the antigen sensitivity of CAR-T cells, we engineered CARs targeting CD19 by including intrinsically disordered regions (IDRs) that promote signaling condensation. The “IDR CARs” triggered enhanced membrane-proximal signaling in the CAR-T synapse, which led to an increased release of cytotoxic factors, a higher killing activity towards low antigen-expressing cancer cells in vitro, and an improved anti-tumor efficacy in vivo. No elevated tonic signaling was observed in IDR CAR-Ts. Together, we demonstrated IDRs as a new tool set to enhance CAR-T cytotoxicity and to broaden CAR-T’s application to low antigen-expressing cancers. Citation Format: Xinyan Zhang, Qian Xiao, Longhui Zeng, Fawzaan Hashmi, Xiaolei Su. Intrinsically disordered regions -induced CAR condensation improves the cytotoxicity of CAR-Ts against low-antigen cancers [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-002.

The phospholipase PLCγ1 is essential for T cell activation. It is frequently mutated in Adult T-cell leukemia/lymphoma (ATLL), Peripheral T-cell lymphomas (PTCL), and Cutaneous T-cell lymphoma (CTCL). However, the molecular mechanisms of PLCγ1 mutation in pathogenesis of these T cell malignancies remains unclear. Liquid-liquid phase separation (LLPS) is an emerging principle in organizing cellular signaling. The dysregulation of LLPS derived by aberrant protein aggregation is progressively implicated as pathological mechanism in tumorigenesis. We previously reported that PLCγ1 structurally promotes LLPS of linker for activation of T cells (LAT) to form condensates in physiological TCR signaling. These lead us to hypothesize that leukemia associated PLCγ1 mutations enhanced TCR signaling and T cell growth by promoting abnormal LLPS. Three frequent and persistent PLCγ1 mutations (R48W, S345F, and D1165H) were chosen in this study. The mutated PLCγ1 recombinant proteins significantly boosted membrane associated LAT condensation compared with wild type (WT) at physiological concentration in a membrane-based biochemical reconstitution system. Increased condensation of LAT was confirmed in live T cells through total internal reflection fluorescence (TIRF) microcopy. Consequently, the downstream signaling including calcium influx and ERK phosphorylation were enhanced in T cell cancer lines harboring PLCγ1 mutations. PLCγ1 mutations also significantly induced the secretion of cytokines and chemokines such as IL-2 and CXCL10. Ectopically expression of PLCγ1 mutants in human primary T cells promotes T cell proliferation, CD69 expression, and effector memory T cell development. Together, these results suggested that leukemia-associated PLCγ1 mutations drive abnormal LLPS to boost TCR signaling and promote T cell proliferation. We also determined if PLCγ1 mutations render any drug resistance by testing a few T cell lymphoma drugs in clinical use. The T cell lymphoma cell line Hut78 expressing PLCγ1 mutations showed resistance to histone deacetylase (HDAC) inhibitors. Moreover, PLCγ1 mutations reduced apoptosis under HDAC inhibition, with a company of enhanced Bcl-2 expression. The Human protein kinase phosphorylation array assay was performed to reveal the mechanism underlying drug resistance. We found that inhibiting MAPK could reverse the HDAC inhibitor resistance at non-cytotoxicity concentrations. These results demonstrated that PLCγ1-MAPK signaling axis confers the HDAC inhibitor resistance. In summary, leukemia-associated PLCγ1 mutations facilitate abnormal LLPS formation to enhance the T cell receptor signaling. Gain-of-function of PLCγ1 mutations cause drug resistance to HDAC inhibitors, which can be rehabilitated by inhibiting the MAPK pathway. Our work reveals molecular mechanisms underlying T cell lymphomagenesis and provides solutions to patients suffering from drug resistance.

One of the long-standing questions in cell signaling field to identify and characterize key signaling nodes out of a complex network. Phospholipase Cγ1 (PLCG1) was identified as the most frequently mutated gene in adult T-cell leukemia/lymphoma, suggesting a critical function of PLCG1 in driving T cell activation. However, it remains unclear how these mutations regulate T cell physiology and pathology. Here we investigated three common leukemia/lymphoma associated mutations (R48W, S345F, and D1165H). We discovered that these mutations induced hyperactive T cell signaling and caused pro-survival phenotypes. PLCG1 mutants enhanced LAT condensation, calcium influx, and ERK activation. They promoted T cell proliferation, induced cell aggregation, and rendered resistance to vorinostat, an FDA-approved drug for cutaneous T-cell lymphoma. The resistance to vorinostat depended on ERK signaling and can be reversed with an ERK inhibitor. Mechanistically, alpha smooth muscle actin, which was specifically induced by PLCG1 mutants, directly bound PLCG1 to promote its activation. Together, these results demonstrated that hyperactive PLCG1 promoted T cell survival and drug resistance through inducing non-canonical signaling.