Biology subjects

Bhatia, S.

Publications and source records attributed to Bhatia, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Toward A Whole-Brain Interface Enabled by High-Resolution Surface Electrophysiology

The goal of building a whole-brain interface - capable of generating a real-time digital representation of the state of the entire central nervous system - has long been framed as a hardware challenge of recording simultaneously from ever-larger numbers of microelectrodes. Advances in high-resolution neural interfaces now enable electrophysiologic interaction with the human nervous system at spatial and temporal resolutions previously unattainable without disrupting tissue architecture or physiology. These capabilities create new opportunities for mechanistic neuroscience, neuroprosthetics, and precision-guided neurosurgery. With the advent of modern microelectrode array technology, however, the whole-brain interface has also become a data challenge. Rather than pursuing large-scale human brain recording purely through a single device placed in a single patient, we demonstrate here that systematic multi-subject aggregation, across brain regions and behavioral paradigms, in a common anatomical framework, can achieve something functionally similar to a whole-brain interface. Here we present an approach to a functional atlas of the human central nervous system derived from high-resolution surface electrophysiology recordings obtained in the first 70 patients implanted with an anatomically conformal, thin-film neural interface positioned in more than 100 locations. The atlas comprises >100,000 electrode deployments and catalogs placement across a broad range of neuroanatomic targets spanning the motor, sensory, language, auditory, and associative domains, as well as brainstem and spinal cord structures that have historically been difficult to access at high electrophysiologic resolution. Across participants, aggregated recordings reveal somatotopic organization, including digit representations that follow a lateral-to-medial ordering consistent with classical homuncular maps, with transitions between adjacent representations occurring over spatial scales of approximately 0.8 - 2.0 mm; canonical frequency-band signatures, such as movement-related beta-band suppression and focal high-gamma (70 - 200 Hz) increases during motor and speech behaviors; and task-modulated population dynamics in both evoked and spontaneous activity across cortical, brainstem, and spinal cord regions. These findings yield insight into patterns of mesoscale electrophysiological dynamics conserved across the entire human central nervous system, suggesting that fine-scale functional organization is preserved across individuals when aligned to common anatomical landmarks. We further demonstrate several applications of these high-resolution recordings, including real-time neural decoding from upper extremity motor cortex to enable real-time neuroprosthetic control, and from ventrolateral cortex to decode cortical correlates of spoken language, facial movements, and complex facial expressions of emotion, supporting initial feasibility of combined verbal and nonverbal communication interfaces. Together, these results establish a paradigm for approaching large-scale interfaces with the human brain, as well as a starting point for a scalable, extensible atlas of human electrophysiologic function designed to accelerate discovery across basic neuroscience, translational research, and clinical neurotechnology.

neuroscience↗

bioRxiv2026

Vulnerability of triple negative breast cancer to daraxonrasib pan-RAS(on) inhibition

Triple-Negative Breast Cancer (TNBC) is a clinical subtype where aberrant expression of receptor tyrosine kinases (RTKs) leads to the activation of the RAS-RAF-MEK-ERK pathway contributing to tumor progression. Therapies targeting components of the RAF-MEK-ERK pathway in TNBC have been disappointing due to rapid onset of adaptive reprogramming of the RTK landscape leading to pathway reactivation. To overcome the dynamic heterogeneity of RTK expression and their convergence in activating RAS, treatment with the pan-RAS(on) inhibitor daraxonrasib (RMC-6236), which inhibits RAS-GTP regulation of RAF kinases, inhibited proliferation independent of TNBC kinotype or oncogenic RAS mutation in three of four TNBC cell lines in vitro and two patient-derived TNBC xenografts in mice. Daraxonrasib in combination with the MEK inhibitor trametinib prevented reactivation of the RAF-MEK-ERK pathway observed with each inhibitor alone and further enhanced inhibition of tumor growth in TNBC cell lines and xenografts. Inhibiting pan-RAS(on) RAS-GTP, with daraxonrasib, alone or in combination with a MEK inhibitor, provides a new strategy for the treatment of TNBC that inhibits RTK activation of the RAS-RAF-MEK-ERK pathway and tumor proliferation.

cancer biology↗

bioRxiv2026

Phenotypic screens identify biologic regulators of nanoparticle uptake in diffuse midline glioma

Nanoparticle drug delivery systems hold considerable promise for locoregional administration to central nervous system tumors, yet the biological determinants of nanoparticle-cancer cell interactions remain poorly understood. Using patient-derived histone-mutant diffuse midline glioma (DMG) models, we performed a pooled CRISPR-Cas9 perturbation screen to systematically identify regulators of liposomal nanoparticle delivery. The screen identified candidate genes spanning endocytosis, vesicle transport, and metabolic signaling, revealing that nanoparticle delivery is governed by a broader landscape than previously appreciated. Among these, CTNNB1, or {beta}-catenin, emerged as a common negative regulator across two independent DMG models and two distinct nanoparticle surface chemistries. Transcriptomic profiling of CTNNB1-depleted DMG cells revealed upregulation of membrane remodeling and extracellular matrix gene programs, accompanied by reduced cell stiffness measured by a microfluidic acoustic scattering assay. This resulted in a shift in endocytic activity characterized by decreased bulk-phase macropinocytosis and increased receptor-mediated endocytosis. We further identified MAPK and mTOR pathway members as nanoparticle trafficking modulators, and demonstrated concordance between genetic and pharmacologic perturbations in modulating the liposomal nanoparticle interactions in pediatric DMG cells. These findings establish a biology-first screening approach for identifying previously unappreciated regulators with potential relevance to nanoparticle-based therapeutic strategies in pediatric brain tumors.

bioengineering↗

bioRxiv2026

Functional evaluation of a natural AAV capsid liver targeting motif in human hepatocytes

Background & Aims: Adeno-associated virus (AAV) vectors are attractive delivery vehicles for therapeutic gene delivery, and a notable feature of most AAVs is their natural tropism for the liver, which leads to significant hepatic uptake following systemic administration. In previous work, we identified 266G as a conserved motif on a variable region on the capsid of many commonly used AAV variants that controls liver uptake in both mice and non-human primates. This single amino acid could be functionally leveraged to engineer AAVs to either de-target from or enhance tropism to the liver. Here, we explored whether these observations extended to the human context. Methods: Two human hepatocyte models were tested: Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice with humanized livers and a bioengineered human microliver platform in vitro. A barcoded AAV capsid library including standard control serotypes were used to assess the role of the 266G motif on gene transfer and transgene expression in both liver systems. Results: In vivo, 266G containing AAVs indeed targeted human hepatocytes superiorly, with some noted dependency on the degree of human-hepatocyte replacement in the chimeric mouse model. Initial studies in the micropatterned primary human hepatocyte co-culture model however demonstrated enrichment of heparin-binding AAVs, and not 266G variants. Notably, incorporation of polyethylene glycol (PEG) into the system modified the AAV transduction potential of those capsids including the liver-targeting motif, recapitulating the hepatocyte transduction pattern observed in vivo. Importantly, when PEG was used, the two human models, both at the DNA and RNA level, did correlate significantly. Conclusions: Our results showed the potential of a combinatorial AAV library for model validation and revealed the human microliver platform-PEG as a reliable system for the development of AAV therapeutics.

molecular biology↗

bioRxiv2026

An immunocompetent Merkel cell carcinoma model for preclinical studies

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin cancer that frequently carries integrated Merkel cell polyomavirus DNA and expresses oncogenic viral small T antigen (sTAg) and truncated large T antigen (tLTAg). We previously reported a mouse model of MCC with skin-targeted expression of sTAg, tLTAg, and the Merkel cell transcription factor ATOH1, combined with deletion of Trp53. Here, we optimized this model to achieve 100% tumor penetrance with lymph node metastases, established four mouse MCC cell lines, and selected one line, mMCC2, for pilot preclinical trials. In immunocompetent C57BL/6J mice, mMCC2 cells reliably produce MCCs and lymph node metastases following subcutaneous or intradermal (orthotopic) injection, and liver and lung metastases after tail vein injection. Mouse MCC allografts resemble parental tumors histologically and express a full complement of MCC differentiation markers. Treatment of allografted mice with anti-PD-1 resulted in variable inhibition of tumor growth. In contrast, treatment with lysine-specific histone Wdemethylase 1 (LSD1) inhibitors, with or without anti-PD-1, led to consistently lower tumor volumes by 5.7-fold in both groups (P < 0.0001) and smaller or undetectable lymph node metastases. Growth-inhibited tumors in all groups showed a marked reduction in proliferating tumor cells and increased infiltration by F4/80+ macrophages and CD8+ T cells. These findings support a role for immune-cell recruitment in treatment response and underscore the importance of immunocompetent preclinical models, even in studies using targeted therapies. This unique virus-positive MCC allograft model, which produces local tumors as well as regional and distant metastases in immunocompetent hosts, provides a critical platform for preclinical evaluation of new therapeutic strategies and sets the stage for much-needed translational studies to inform future clinical trials.

Cancer Biology↗

bioRxiv2026

Patient-derived organoid xenografts reveal the multifaceted role of the lncRNA MALAT1 in breast cancer progression

Long non-coding RNAs (lncRNAs) have emerged as key regulators of tumor biology, however, thus far none have translated to cancer therapies. The lncRNA MALAT1 is overexpressed in more than 20 cancers, including breast cancer and has been shown to function via various mechanisms in a context-dependent manner, in 2D cell lines and mouse models. However, its functional role and therapeutic potential have not been evaluated in clinically relevant patient-derived models. We investigated the therapeutic potential of MALAT1-targeting antisense oligonucleotides (ASOs) for breast cancer, using clinically relevant 3D human patient-derived organoids (PDOs) and PDO-xenograft (PDO-X) models. We systematically evaluated the efficiency of MALAT1-targeting ASOs using a biobank of 28 PDO models. Across three independent PDO-X models of triple negative breast cancer (TNBC), MALAT1 depletion reproducibly drove widespread alternative splicing changes across all event types, with an enrichment of intron retention. Differentially spliced transcripts were enriched for targets of shared cancer-associated transcription factors, and MALAT1 knockdown specifically altered the relative abundance of previously unannotated splicing isoforms. Beyond tumor-intrinsic effects, tumor-specific MALAT1 depletion induced a consistent reduction in macrophage-associated gene signatures and reduced lung metastatic burden. Our data defines MALAT1s multifaceted role in TNBC, coordinating alternative splicing, tumor-stroma crosstalk, and metastatic progression. Our study provides strong preclinical evidence supporting MALAT1-targeted ASO therapy and establishes PDO-X models as a clinically relevant platform for functional interrogation of TNBC therapies. SignificanceUsing clinically relevant human PDO-X models, we show that MALAT1 influences alternative splicing, metastatic potential and the tumor microenvironment. These data support the potential of oncogenic lncRNA MALAT1 as a therapeutic target.

cancer biology↗

bioRxiv2025

Synthetic Control of Implanted Engineered Liver Tissue Growth

Despite the promise of engineered tissue implants for the treatment of organ failure, scaling of these constructs to sizes of therapeutic relevance remains a barrier to clinical translation. Here, we propose a strategy to circumvent this limitation: to instead implant a small-scale construct and then induce it to grow in situ after its engraftment into a host. Using engineered liver tissue as a proof-of-concept application, we integrated synthetic biology and tissue engineering tools to build liver tissues that can be expanded on-demand after implantation in vivo. To achieve this goal, we first identified the combination of YAP and growth factor signaling as sufficient to drive human hepatocyte proliferation in dense, 3D engineered tissues. We then engineered control of these signaling axes using synthetic biology tools to drive human liver tissue expansion both in vitro and in vivo. As such, this work establishes a genetic strategy for generating large organ implants through bioengineered, on-demand outgrowth using synthetic triggers (BOOST). TeaserPresenting bioengineered on-demand outgrowth via synthetic biology triggering (BOOST) for in situ solid cell therapy scale up.

bioengineering↗

bioRxiv2025

Enhancer-directed gene delivery for digit regeneration based on conserved epidermal factors

Limb loss remains a significant clinical challenge, but regenerative medicine approaches such as gene therapy offer a promising strategy to trigger endogenous regeneration programs. Optimal vector configurations and molecular targets for appendicular skeletal repair are not well defined. Here, we leveraged insights from species with a high endogenous capacity for appendage regeneration to design an enhancer-directed gene delivery platform that functions during mouse digit regeneration, a well characterized model for partial limb regeneration in mammals. Single-cell RNA sequencing of zebrafish caudal fin regeneration, combined with expression data in regenerating salamander limbs and mouse digit tips, implicated the SP family of transcription factors as conserved, epidermally-expressed mediators of appendage regrowth. Null mutants of Sp8 demonstrated impaired limb regeneration in salamanders, while conditional knockout of Sp6 and/or Sp8 in the mouse basal epidermis resulted in defective bony digit tip regeneration, involving an IL-17 mediated osteoclastogenic program. Spatiotemporally focused expression of FGF8, a known target of SP factors, using a zebrafish-derived tissue regeneration enhancer element via adeno-associated viral vectors, could partially rescue digit tip regeneration in SP knockout mice and accelerate digit regeneration in wildtype mice. Our results demonstrate a contextual gene therapy approach to address limb loss based on genes like SP transcription factors conserved across multiple contexts of appendage regeneration. Significance StatementInstructing regeneration of complex structures in mammals remains an unsolved problem. Gene therapy offers a compelling approach to foster endogenous regeneration by delivering therapeutic gene products to specific cells post injury. We identified a conserved regeneration-linked epidermal transcriptional program in mouse digit regeneration centered on the SP6 and SP8 transcription factors, involving inflammatory responses from osteoclasts. We engineered AAVs harboring a zebrafish tissue regeneration enhancer to direct FGF8 expression in the epidermis after amputation. This enhancer directed delivery partially rescued impaired digit regeneration in Sp6 and Sp8 conditional knockout mice and accelerated regrowth in wildtype digits. Our work links developmental signaling to adult regeneration and establishes a modular, injury site specific gene therapy framework that enables new interventions for limb healing.

genetics↗

bioRxiv2025

On-target toxicity limits the efficacy of CDK11 inhibition against cancers with 1p36 deletions

The cyclin-dependent kinase CDK11 is an understudied kinase that has been the subject of conflicting reports regarding its function in cancer. Here, we combine genetic and pharmacological approaches to demonstrate that CDK11 is a critical regulator of cancer cell survival that is required for RNA splicing and the expression of homologous recombination genes. Inhibition of CDK11 disrupts genome stability, promotes the retention of intronic sequences in mature mRNAs, and induces synthetic lethality with PARP inhibitors. Through integrative analysis of functional genomics datasets, we identify heterozygous deletions of chromosome 1p36 - which encompasses CDK11 and its activating cyclin CCNL2 - as a recurrent and predictive biomarker of sensitivity to CDK11 inhibition. To assess the therapeutic potential of CDK11, we develop MEL-495R, a selective and orally bioavailable CDK11 inhibitor. Additionally, we establish a genetically-engineered mouse model that allows us to differentiate between the on-target and off-target effects of CDK11 inhibitors in vivo. Using this platform, we demonstrate that MEL-495R induces widespread on-target toxicity, revealing a narrow therapeutic index. Together, these findings define CDK11 as a core cancer dependency, uncover a chromosomal deletion that sensitizes tumors to CDK11 inhibition, and provide a generalizable strategy for deconvolving drug efficacy and toxicity in vivo for novel oncology targets.

cancer biology↗

bioRxiv2025

Multimodal profiling of proinflammatory protease activity identifies caspase-1 as a target for lung cancer interception

The systemic inhibition of IL-1b, a key mediator of pulmonary inflammation, has been shown to reduce the incidence of lung cancer in patients in years following treatment, but knowledge gaps surrounding its activation and role in the tumor microenvironment are hindering approaches for cancer interception. We developed a suite of activity-based technologies to probe inflammation in early lung cancer and identified a translational target candidate. We designed probes sensitive to various IL-1b-activating proteases and applied them to a murine model of inflammatory lung cancer, Kras/Trp53-mutant with SIINFEKL expression (KPS). Our nanosensors revealed reduced cleavage of a caspase-1 reporter in the lungs of KPS mice treated with IL-1b antibody, as well as elevated caspase-1 expression and activity in naive tumor tissue sections, highlighting the importance of caspase-1 processing of IL-1b during cancer development. We conducted a pre-clinical trial of a novel combination intervention by administering both IL-1b blockade and caspase-1 inhibition shortly after tumor induction. Following treatment, we observed significant reduction in lung cancer formation, including complete ablation of tumor incidence in nearly 20% of KPS mice. Our approach to understand the interplay of protease activity and cytokine activation supports development of new strategies to mitigate inflammation and intercept lung cancer progression.

bioengineering↗

bioRxiv2025

Epigenetic remodeling via HDAC6 inhibition amplifies anti-tumoral immune responses in pediatric AML

Histone deacetylase 6 (HDAC6) has emerged as a promising therapeutic target in cancer due to its immunomodulatory effects. While its prognostic significance remains debated, we demonstrate that HDAC6 loss significantly impairs myeloid leukemia progression in vivo, despite having no functional impact on leukemia cell proliferation in vitro. Global proteome and secretome profiling of HDAC6-knockout (KO) cells revealed upregulation of several immune-related modulators, including RNase T2, a tumor suppressor known to modulate the tumor microenvironment. Notably, RNase T2 upregulation upon HDAC6 loss was restricted to myeloid but not B-ALL cells. Moreover, pharmacological inhibition of HDAC6 recapitulated this phenotype, leading to RNase T2 upregulation in myeloid leukemia cells. ATAC-seq revealed increased chromatin accessibility of RNase T2 following HDAC6 loss, highlighting a functionally epigenetic regulatory contribution. Further functional assays conducted in an immunocompetent setting both ex vivo and in vivo demonstrated that HDAC6 inhibition sensitized murine myeloid leukemia cells to broad CD8+ T cell activation as evidenced by increased TNF and CD107a expression. Consistently, in a syngeneic model, HDAC6 inhibition restricted growth myeloid leukemia cells. Moreover, an extended drug screening analysis identified Cytarabine and Clofarabine as significantly synergizing with HDAC6 inhibitor (Ricolinostat) in myeloid leukemia cell lines and in patient derived xenograft (PDX) cells, while showing limited synergy in lymphoid leukemia cell lines, PDX or healthy control cells. These findings suggest that HDAC6 represents a promising therapeutic target in myeloid lineage derived leukemia cells by simultaneously enhancing immune activation and increasing chemosensitivity.

cell biology↗

bioRxiv2025

Highly Pathogenic Avian Influenza A (H5N1) Clade 2.3.2.1a virus infection in domestic cats, India, 2025

In January 2025, the highly pathogenic avian influenza A(H5N1) virus clade 2.3.2.1a infection was detected in domestic cats and whole-genome sequencing of two cat H5N1 isolates was performed using the Oxford Nanopore MinION sequencing platform. Phylogenetic analysis revealed the circulation of triple reassortant viruses in cats. Although cat viruses lacked classic mammalian adaptation markers they carried mutations associated with enhanced polymerase activity in mammalian cells and increased affinity for 2-6 sialic acid receptor suggesting their potential role in facilitating infection in cats. The identification of reassortant HPAI H5N1 clade 2.3.2.1a viruses in domestic cats in India highlights the urgent need for enhanced surveillance in domestic poultry, wild birds, and mammals, including humans, to track genomic diversity and molecular evolution of circulating strains.

microbiology↗

bioRxiv2025

Neuronal Silencing and Protection in a Mouse Model of Demyelination

Damage to the myelin sheath that protects axons in the central nervous system is a hallmark pathology of demyelinating diseases like multiple sclerosis. Cuprizone-induced demyelination in mice is a common model for studying demyelination and remyelination. However, the relationship between myelin damage and recovery and neuronal function remains poorly understood. By monitoring hippocampal myelination and neuronal activity in the same mice, we assessed longitudinal changes following cuprizone consumption and remyelination treatment. Upon cuprizone consumption, a rapid decline in neuronal activity preceded slower demyelination. Female mice showed a more intense early decrease in brain activity and a stronger correlation with levels of myelin loss. Remyelination treatment led to increased myelination levels and recovery of neuronal activity compared to vehicle treatment. Changes in single-neuron firing rates during treatment were proportional to the pre-cuprizone firing rate in the same cells, highlighting a potential linkage between the status of myelin recovery and cellular activity.

neuroscience↗

bioRxiv2024

Expandable, Functional Hepatocytes Derived from Primary Cells Enable Liver Therapeutics

Liver disease affects millions annually in the United States, with orthotopic transplantation as the only curative option for many patients. However, the scarcity of donor organs highlights a need for alternative cell-based therapies. Hepatocyte-based approaches are promising due to the cells inherent synthetic, metabolic, and detoxifying functions, but they face critical barriers, including the lack of a scalable source of functional hepatocytes and poor engraftment. In this study, we developed a scalable process for expanding primary human hepatocytes (PHHs) while preserving their identity and function. By leveraging heterocellular aggregation with stromal cells, we generated cryopreserved "seed" constructs that maintained viability and function post-thaw. Seeds demonstrated enhanced metabolic and detoxification functions and robust engraftment across multiple anatomic sites outside of the liver. Our approach addresses key limitations of hepatocyte-based therapies, offering a stable, scalable, and clinically viable platform for liver cell therapy applications.

bioengineering↗

bioRxiv2024

Conditional fusogenic lipid nanocarriers for cytosolic delivery of macromolecular therapeutics

Macromolecular therapeutics designed for intracellular targets must overcome systemic delivery barriers, target cell membrane impermeability, and inefficient endosomal escape. Here, we engineer a class of conditional fusogenic liposomes (C-FLIPs) that harness the catalytic activity of extracellular proteases present in the pathological microenvironment. This context-specific activation enables on-target membrane fusion with cells in diseased tissue, resulting in cytosolic delivery of therapeutic payloads. We describe the cytoplasmic delivery of three prototypic macromolecular therapeutic classes: peptide degraders, cytotoxic proteins, and ribonucleoprotein particles (RNPs). We further develop C-FLIP to deliver granzyme B (GzmB) to the cytoplasm of cancer cells in vivo and induce pyroptosis in immunologically-inert tumors. Treatment with C-FLIP/GzmB reprograms the immunosuppressive tumor microenvironment and synergizes with checkpoint blockade to result in the regression of established tumors and induce immunological memory. This modular, non-viral, cytosolic delivery platform represents a promising approach to leverage pathological protease activity for targeted delivery of biologics.

bioengineering↗

bioRxiv2024

Small-molecule inhibitor of C-terminal HSP90 dimerization modulates autophagy and functions synergistically with mTOR inhibition to kill cisplatin-resistant cancer cells

BackgroundA major obstacle for the successful treatment of cancer is the primary presence or development of resistance mechanisms toward therapeutic intervention. In urothelial cancer, cisplatin-based regimens are still routinely employed, and multiple cellular pathways contribute to chemoresistance. Since the identification of heat shock protein 90 (HSP90) as potential cancer target, various HSP90 inhibitors (HSP90i) have been developed and evaluated in clinical trials. However, limited efficacy has been observed, mainly caused by dose-limiting toxicity and the concomitant induction of a cytoprotective heat shock response (HSR). To avoid this effect, inhibitors targeting the C-terminal domain (CTD) of HSP90 that do not elicit an HSR have been put forward. Additionally, the crosstalk between autophagy and HSP90 is currently being explored, since both processes work together in proteostasis, and the modulation of autophagic responses might be helpful in order to improve the efficacy of HSP90 inhibitors. MethodsThe second-generation small-molecule inhibitor VWK147 targeting HSP90 CTD dimerization was synthesized and characterized in detail by biochemical cell-free and cellular assays and molecular modeling. Specifically, HSP90 inhibition, cell viability, and autophagy were monitored in mono- and combined treatments. ResultsWe demonstrate that VWK147 induces cell death in both cisplatin-sensitive and cisplatin-resistant urothelial carcinoma cells. The treatment with VWK147 in these cells led to the destabilization of classical HSP90 client proteins without triggering an HSR. Additionally, we observe that VWK147 re-sensitizes resistant urothelial carcinoma cells to cisplatin and--in combination with mTOR inhibition--synergistically kills cisplatin-sensitive and -resistant cells, in contrast to what is observed upon treatment with the N-terminal domain-targeting HSP90 inhibitor 17-AAG. This synergy may be explained by VWK147-mediated inhibition of late autophagy events, and thus a blockade of autophagic flux. Finally, we also observed that VWK147 induces non-canonical LC3 lipidation, indicating that this compound possibly exerts a broader effect on ion balance or pH of the endolysosomal system. ConclusionVWK147 is a promising inhibitor that targets the C-terminal dimerization of HSP90 and simultaneously exhibits autophagy-modulating effects. This compound could potentially be an effective option for improving anti-cancer therapies and/or overcoming treatment resistance.

cell biology↗

bioRxiv2024

Quantitative proteomic mass spectrometry of protein kinases to determine dynamic heterogeneity of the human kinome

The kinome is a dynamic system of kinases regulating signaling networks in cells and dysfunction of protein kinases contributes to many diseases. Regulation of the protein expression of kinases alters cellular responses to environmental changes and perturbations. We configured a library of 672 proteotypic peptides to quantify >300 kinases in a single LC-MS experiment using ten micrograms protein from human tissues including biopsies. This enables absolute quantitation of kinase protein abundance at attomole-femtomole expression levels, requiring no kinase enrichment and less than ten micrograms of starting protein from flash-frozen and formalin fixed paraffin embedded tissues. Breast cancer biopsies, organoids, and cell lines were analyzed using the SureQuant method, demonstrating the heterogeneity of kinase protein expression across and within breast cancer clinical subtypes. Kinome quantitation was coupled with nanoscale phosphoproteomics, providing a feasible method for novel clinical diagnosis and understanding of patient kinome responses to treatment.

biochemistry↗

bioRxiv2024

The pro-tumoral and anti-tumoral roles of EphA4 on T regulatory cells and tumor associated macrophages during HNSCC tumor progression.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a deadly cancer with poor response to targeted therapy, largely driven by an immunosuppressive tumor microenvironment (TME). Here we examine the immune-modulatory role of the receptor tyrosine kinase EphA4 in HNSCC progression. Within the TME, EphA4 is primarily expressed on regulatory T cells (Tregs) and macrophages. In contrast ephrinB2, an activating ligand of EphA4, is expressed in tumor blood vessels. Using genetically engineered mouse models, we show that EphA4 expressed in Tregs promotes tumor growth, whereas EphA4 expressed in monocytes inhibits tumor growth. In contrast, ephrinB2 knockout in blood vessels reduces both intratumoral Tregs and macrophages. A novel specific EphA4 inhibitor, APY-d3-PEG4, reverses the accelerated tumor growth we had previously reported with EphB4 cancer cell knockout. EphA4 knockout in macrophages not only enhanced their differentiation into M2 macrophage but also increased Treg suppressive activity. APY-d3-PEG4 reversed the accelerated growth seen in the EphA4 knockout of monocytes but conferred no additional benefit when EphA4 was knocked out on Tregs. Underscoring an EphA4-mediated interplay between Tregs and macrophages, we found that knockout of EphA4 in Tregs not only decreases their activation but also reduces tumor infiltration of pro-tumoral M2 macrophages. These data identify Tregs as a primary target of APY-d3-PEG4 and suggest a role for Tregs in regulating macrophage conversion. These data also support the possible anti-cancer therapeutic value of bispecific peptides or antibodies capable of promoting EphA4 blockade in Tregs but not macrophages. SignificanceEphA4 in regulatory T cells has a pro-tumoral effect while EphA4 in macrophages plays an anti-tumoral role underscoring the necessity of developing biologically rational therapeutics.

cancer biology↗