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Choudhary, A.

Publications and source records attributed to Choudhary, A..

At least 19 recordsLinked to original sources

Jasmonate-induced expansion of the MYC2 cistrome occurs within a preconfigured chromatin landscape

The transcription factor (TF) MYC2 is a central regulator of jasmonate (JA) signaling, yet its genome-wide occupancy has primarily been characterized using transgenic lines expressing tagged MYC2 proteins, leaving the dynamics of the native MYC2 cistrome unresolved. Here, using a MYC2-specific antibody, we define the wildtype MYC2 cistrome in Arabidopsis thaliana under basal and JA-induced conditions. Native MYC2 showed minimal genome-wide occupancy under control conditions but underwent a pronounced expansion following JA treatment, resulting in more than 3,000 binding sites enriched at core components of the JA signaling network. Cistrome expansion coincided with increased MYC2 protein abundance, while elevated basal MYC2 levels in a transgenic line were associated with extensive MYC2 occupancy even in the absence of JA, supporting a dosage-dependent relationship between MYC2 abundance and genome-wide binding. Despite this extensive cistrome expansion and transcriptional reprogramming, ATAC-seq revealed remarkably limited remodeling of chromatin accessibility following JA treatment. MYC2 recruitment occurred predominantly within pre-existing accessible chromatin regions that remained largely intact in myc2 myc3 myc4 mutants. Together, our findings reveal that JA-dependent expansion of the native MYC2 cistrome occurs within a largely pre-established accessome and identify MYC2 dosage as an important determinant of the extent of genome-wide occupancy.

plant biology↗

In-Silico Thermodynamic and Structural Profiling of Bacterial SoxB Thiohydrolase: Evaluating the Substrate Accommodation of Circular Potassium Thiosulfate in Sulfur-Deficient Alkaline Soils

Characterizing the enzymatic accommodation of circular agricultural fertilizers is critical for informing strategies to remediate widespread soil sulfur hunger. Here, we present an exploratory in-silico investigation evaluating the active-site cleft of sulfate thiohydrolase (SoxB) across representative soil Proteobacteria. Following the crystallographic precedent of uncomplexed thiosulfate in PDB 2WDE, site-directed molecular docking indicated that the free thiosulfate polyanion binds favorably within the catalytic pocket (predicted affinity: -3.506 kcal/mol), yielding a substantially lower empirical energy barrier than hydrophobic elemental sulfur (S8, -1.409 kcal/mol). Comparative evaluation against the alkaline-adapted Thiobacillus denitrificans homolog revealed an elevated predicted binding affinity of -4.610 kcal/mol, suggesting a potential structural accommodation in high-pH calcareous soils (pH > 8.0). Unrestrained 5.0 ns all-atom molecular dynamics in explicit TIP3P solvent demonstrated initial structural stability of the unliganded host backbone (RMSD = 1.10 +/- 0.15 Angstrom). Energetic decomposition indicated that ligand association is governed predominantly by electrostatic interactions (delta-E_elec ~ -40 kcal/mol) with basic residues (His146, His269, Trp147; RMSF < 0.60 Angstrom), with a single-trajectory unbinding event observed at 3.2 ns. These computational observations provide an exploratory baseline characterizing the active-site electrostatic landscape of SoxB, generating working hypotheses for downstream empirical soil microcosm and in-planta trials.

bioinformatics↗

KMT2A modulates the epigenetic landscape of rDNA by facilitating the recruitment of histone lysine acetyltransferase PCAF to the rDNA locus.

Histone acetylation is often associated with transcriptional activation across a wide range of genes, playing a key role in RNA Polymerase II dynamics. However, its specific role in transcriptional activation of RNA Polymerase I (RNA Pol I) remains unclear. In this study, we demonstrate that KMT2A associates with ribosomal DNA (rDNA) loci. Notably, the loss of KMT2A in our inducible KO cell line does not affect the levels of H3K4me3 on rDNA. We support these observations with analyses of ChIP-seq data from mouse embryonic stem cells. While H3K4 methylation remains unchanged, the absence of KMT2A causes a significant decrease in H3 acetylation levels on rDNA, especially in H3K9 acetylation levels. To identify the histone lysine acetyl transferases (KATs) that cooperate with KMT2A in promoting rDNA transcription, we examined the occupancy of multiple KATs and their associated histone acetylation marks on the rDNA locus. Our analyses identify PCAF (p300/CBP associated factor) as the KAT that contributes to KMT2A-mediated transcriptional activation of rDNA. Depletion of KMT2A reduces the levels of PCAF on rDNA, suggesting that KMT2A plays the role of a co-activator of RNA Pol I in the recruitment of KATs on the rDNA loci. Finally, the depletion of KMT2A leads to the disruption of the pre-initiation complex from the rDNA 47S promoter, resulting in a stalled RNA Pol I complex at the spacer promoter. Our findings elucidate the non-redundant, distinct function of KMT2A in the regulation of RNA Pol I transcription.

molecular biology↗

Dynamic histone hyperacetylation shapes environmentally responsive chromatin states

Transcription factors (TFs) orchestrate environmental responses by activating target genes, yet how they reshape epigenome architecture to coordinate gene expression remains poorly understood. We previously identified SIENA (Stimulus-Induced ENhancer Acetylation) domains as large regions of jasmonic acid (JA)-induced H3K9 hyperacetylation surrounding MYC2 TF binding sites in Arabidopsis and tomato. However, the mechanisms underlying the formation of SIENA domains (SIENAs) and their functional significance remained unknown. Here, we show that SIENAs also form at major JA-responsive genes and gene clusters in soybean, extending this phenomenon to an evolutionarily distant crop species. Comprehensive chromatin profiling revealed that SIENAs accumulate multiple histone acetylation marks, including H3K9ac, H3K27ac, H3K56ac, H2BK20ac, and H2A.Zac, establishing them as regions of broad histone hyperacetylation. Pharmacological disruption of proteasomal turnover and histone acetylation dynamics compromised SIENA formation. Chromatin accessibility analyses further showed that inducible accessibility within SIENAs is tightly associated with MYC2 binding sites, supporting a model in which MYCs nucleate localized chromatin reprogramming events. Together, our findings establish SIENAs as MYC2-dependent chromatin-organizing domains and identify histone hyperacetylation as a central feature of MYC2-mediated gene activation.

Plant Biology↗

Polysome Profiling Method for Low-Input Human Postmortem Brain

Polysome profiling is a powerful technique used to analyze the association of mRNA with ribosomes, providing insights into the translational status of a cell. It relies on the separation of ribosome-bound mRNAs through sucrose density gradient centrifugation, where the number of ribosomes on an mRNA correlates with its sedimentation rate. While numerous studies have successfully applied this method to cell line and mouse tissue, application to the human postmortem brain remains scarce due to challenges related to sample quality and low concentration of recoverable material. To overcome these challenges, we: O_LIImplemented a protocol specifically optimized for low-concentration human post-mortem brain tissue. C_LIO_LIImplemented a gradient-maker-free method to manually prepare sucrose gradients with tunable sensitivity for low-input samples. C_LIO_LIAdapted the human brain tissue protocol for neuronal cell lines and mouse brain with minimal modification. C_LI

neuroscience↗

iPSC-derived extracellular vesicles rescue deficits in human and mouse models of Parkinsons disease

Parkinsons disease (PD) pathogenesis often involves progressive -synuclein (-Syn)-mediated neuronal dysfunction, yet the earliest cellular events that link -Syn pathology to circuit failure remain poorly defined. Here, we used human induced pluripotent stem cell (iPSC)-derived dopaminergic (DA) neurons from patients carrying the familial A53T SNCA mutation to reconstruct a temporal course of dysfunction in vitro. We identified a biphasic trajectory with an early phase of hyperexcitability, characterized by elevated spontaneous firing, followed by a progressive transition into hypoexcitability as the neurons mature, accompanied by reduced network activity, synaptic dysfunction, and -Syn accumulation. Transcriptomic profiling at the critical transition point revealed a dual transcriptional signature, with upregulation of stress-inflammatory pathways (p53, JAK-STAT, apoptosis) coupled with systematic downregulation of metabolic and synaptic maintenance genes. This molecular profile preceded functional collapse, linking early hyperactivity-driven metabolic stress to subsequent neuronal exhaustion. To counteract this pathology, we used extracellular vesicles (EVs), small membrane-bound particles carrying intercellular signals, as a cell-free treatment approach. Strikingly, treatment with EVs derived from healthy iPSCs completely rescued both electrophysiological deficits and pathological -Syn accumulation, restoring normal firing patterns, synaptic function, and network activity. Consistent with these observations, EV treatment reduced -Syn aggregation and improved motor responses in -Syn fibril-injected mice, which are characterized by pathological -Syn accumulation and motor deficits. Overall, these findings demonstrate that EVs derived from healthy iPSCs can reverse PD-related phenotypes in human and mouse models.

neuroscience↗

A Scalable Design for Proximity-Inducing Molecules

Chimeric molecules, which bring together an effector enzyme and a protein-of-interest (POI) to add/remove post-translational modifications (PTMs), are furnishing transformative modalities (e.g., PROTACs). However, these chimeras scalability is limited as they employ rare, non-inhibitory binders of effectors. We report GRoup-transfer chimeras for Inducing Proximity (GRIPs) that employ abundantly available effectors inhibitors to append POI binder on the effector using group-transfer handles. To demonstrate scalability, we develop 6 GRIPs classes for 3 PTMs utilizing diverse inhibitor, spanning 16 effector-POI pairs. Furthermore, we report a toolbox of 42 tunable group-transfer handles for Cys/Lys residues and [~]5000 inhibitor-residue pairs for diverse effectors. Using global proteomics, we confirm the specificity for group transfer and PTM editing. GRIPs endowed new functionalities to POI drugs, including preventing rebound signaling upon drug withdrawal, a more potent/persistent inhibition, and inhibitor-induced pathway activation in 4 fully-endogenous systems. In diverse hemi-endogenous systems (tagged POI), GRIPs induced condensate formation with reduced off-targets, cleared pathogenic PTMs, and initiated PTM crosstalk. Overall, GRIPs provide a scalable and versatile platform for PTM editing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/706349v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@132f70dorg.highwire.dtl.DTLVardef@f9aa62org.highwire.dtl.DTLVardef@12143edorg.highwire.dtl.DTLVardef@f296d4_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

Anti-CRISPR-mediated continuous directed evolution of CRISPR-Cas9 in human cells

Engineering CRISPR-Cas systems for improved or altered function is central to both research and therapeutic applications. Unfortunately most optimization, especially directed evolution in bacterial hosts, fails to capture the functional requirements of the complex mammalian cellular milieu, where activity is usually required. Robust strategies to enable continuous directed evolution of genome-targeting agents directly in human cells remain lacking. Here, we introduce CRISPR-MACE (Mammalian cell-enabled Adenovirus-assisted Continuous Evolution) as a foundational technology to address this need. CRISPR-MACE integrates virus-based continuous evolution with anti-CRISPR-based tunable selection to generate novel Streptococcus pyogenes Cas9 variants with both increased and decreased DNA binding capacity and nearly 1000-fold-enhanced resistance to AcrIIA4, the strongest known inhibitor of SpCas9. Notably, across independent evolution campaigns the same Cas9 gatekeeper mutation reproducibly emerged first, enabling subsequent adaptive steps along two interdependent axes of Cas9 function. In addition to advancing CRISPR technologies, this work establishes key principles and synthetic circuits for continuously evolving CRISPR-Cas systems directly in human cells. SIGNIFICANCE STATEMENTCRISPR technologies are typically engineered in bacteria, even though they must function in the far more complex environment of human cells. This gap has limited the discovery of variants with improved DNA recognition or with resistance to inhibitors that operate differently in mammalian systems. Here we establish CRISPR-MACE, a continuous evolution platform that leverages pressure from anti-CRISPR proteins to select Cas9 variants directly in human cells that have novel functions. Evolved variants show improvements in DNA binding strength and residence time, as well as striking escape from the potent Cas9 inhibitor AcrIIA4. Many anti-CRISPR proteins use distinct mechanisms, so our strategy can drive future continuous evolution campaigns in mammalian cells that expand the functional properties of genome-targeting agents.

synthetic biology↗

Nuclear Localization Signals Enable the Cellular Delivery of an Anti-CRISPR Protein to Control Genome Editing

Precise regulation of Cas9 activity is essential to minimize off-target effects, mosaicism, chromosomal alterations, immunogenicity, and genotoxicity in genome editing. Although type II anti-CRISPR proteins (Acrs) can inhibit and regulate Cas9, their size and anionic charge generally prevent them from crossing the cell membrane. Existing Acr delivery methods employing vectors or electroporation are either slow and persistent or require external equipment, limiting their therapeutic utility. To address these challenges, we developed a cell-permeable Acr (6xNLS-Acr), which uses nuclear localization signals (NLSs) to cross the cell membrane. We conjugated 6xNLS-Acr to a fluorescent dye to elucidate its cellular entry mechanism and directly visualized its binding to a fluorescent Cas9{middle dot}gRNA complex to study its inhibitory mechanism. 6xNLS-Acr (IC50 = 0.47 {micro}M) directly transduces human cells, including immortalized cell lines, embryonic stem cells, and 3D cell cultures, within 5 min, inhibiting up to 99% of Cas9 activity and increasing genome-editing specificity by nearly 100%. We further compared 6xNLS-Acr with our anthrax-derived Acr delivery platform. Our results demonstrate that 6xNLS-Acr is the most efficacious cell-permeable CRISPR-Cas inhibitor, significantly enhancing the precision and therapeutic potential of CRISPR-based genome editing.

genomics↗

Divergent Excitability of GABAergic Neurons Derived from Bipolar Disorder Patients Shapes Energy Shifts of Network Dynamics, possibly mimicking mania and depression

Bipolar disorder (BD) is characterized by fluctuating mood states, yet the cellular and circuit-level mechanisms distinguishing lithium-responsive (LR) from non-responsive (NR) patients remain elusive. We derived dentate gyrus granule neurons and GABAergic interneurons from induced pluripotent stem cells (iPSCs) of BD patients stratified by lithium response, and from healthy controls. Using patch-clamp electrophysiology, we assessed intrinsic excitability. We further developed a computational model simulating large-scale neuronal networks based on patient-derived electrophysiological properties and ion channel conductance distributions. Granule neurons from both LR and NR patients exhibited hyperexcitability compared to controls. However, GABAergic neurons showed a striking divergence: LR neurons were hyperexcitable, while NR neurons were hypoexcitable. Transcriptomic profiling revealed distinct molecular signatures between NR and LR neurons, including dysregulation of GABA receptor genes (GABRR1). Computational simulations over 10,000 iterations, mimicking long-term network activity, revealed that dentate granule neuron changes alone failed to recapitulate netwrok shifts between global hyperexcitability and global hypoexcitability. Networks incorporating only granule neuron phenotypes entered persistent hyper- or hypoactive states, depending on lithium response. Remarkably, when GABAergic neuron phenotypes were added to the model, both LR and NR networks exhibited spontaneous transitions between high and low activity states, that may be associated with the mood episodes that the patients exhibit. Control networks did not show such bistability. We, therefore, conclude that GABAergic neuronal excitability is a key determinant of lithium responsiveness in BD and critically shapes the emergence of state-shift dynamics in neural networks. These findings suggest that restoring excitatory/inhibitory (E/I) balance via targeted modulation of interneuron function may offer novel therapeutic avenues for BD.

neuroscience↗

Identification of novel exosomal miRNAs and their role in diagnosis and prognosis of Triple Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a clinically aggressive subtype with poor prognosis and limited treatment options. Exosomal microRNAs (miRNAs), encapsulated within secretory vesicles, have emerged as promising biomarkers for cancer detection and monitoring. In this study, we identify five novel exosomal miRNAs--hsa-miR-6803, hsa-miR-1180, hsa-miR-4728, hsa-miR-1915, and hsa-miR-940--that are consistently overexpressed in TNBC cells, stem-like subpopulations, and patient tumor tissues. Integrated analysis of public datasets and in vitro validation revealed that elevated expression of these miRNAs correlates with poor overall survival. Functional assays demonstrated that miR-1180 and miR-4728 significantly promote TNBC cell migration and invasion. These miRNAs also target critical oncogenic pathways, including Wnt, Notch, and EGFR. Their enrichment in exosomes highlights their translational potential as liquid biopsy-based biomarkers and therapeutic targets. This work is the first to link this miRNA panel to both TNBC tumorigenesis and stem-like cell biology, offering new insights into disease progression and potential strategies for personalized care.

cancer biology↗

Phosphorylation-inducing chimera rewires oncogenic kinase to trigger apoptosis

The active sites electric field is integral to enzymatic catalysis (e.g., substrate recognition) and nature employs charge-altering post-translational modifications (e.g., phosphorylation) to perturb this electric field and regulate enzymes. A chromosomal translocation converts Abelson kinase (ABL) to BCR-ABL, whose hyperactivity drives several cancers. Here, we developed a small molecule, BRD8833, that induces BCR-ABL phosphorylation, which perturbs its active sites electric field with loss of hyperactivity. Unlike "occupancy-driven" inhibitors that require stoichiometric concentrations, BRD8833 operates through an event-driven, substoichiometric mechanism by inducing proximity between two BCR-ABL molecules to trigger the inhibitory phosphorylation and selective apoptosis of BCR-ABL-dependent cancer cells. Furthermore, BRD8833 is effective against other oncogenic ABL fusions or clinically observed resistance mutations, including those to occupancy-driven drugs with the same binding site as BRD8833, suggesting differences in their resistance mechanisms. These studies lay the foundation for electric-field and "event-driven" modalities to control hyperactive enzymes with orthogonal resistance mechanisms to occupancy-driven drugs. GRAPHICAL ABSTRACT (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/659082v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1607a40org.highwire.dtl.DTLVardef@fa10deorg.highwire.dtl.DTLVardef@1698232org.highwire.dtl.DTLVardef@1e2e0f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

Orthogonal resistance mechanisms of classical- and induced-proximity inhibitors

Resistance development is an inevitable failure mode of many drugs, pointing to the need to develop agents with orthogonal resistance mechanisms. Induced-proximity modalities, an emergent class of therapeutics, operate by forming a ternary complex with the protein-of-interest (POI) and effectors, unlike classical inhibitors that form binary complexes with the POI. Using KRAS as a model system, we employed base editor tiling mutagenesis screening to show that induced-proximity inhibitors exhibit orthogonal resistance mechanisms to classical inhibitors despite overlapping binding sites, offering an opportunity to circumvent resistance mechanisms of classical inhibitors. These findings highlight the use of base editor mutagenesis screens to prioritize inhibitors with orthogonal resistance mechanisms and the potential of induced-proximity inhibitors to overcome the drug resistance of classical inhibitors.

biochemistry↗

Phosphorylation-inducing molecules for regulating dynamic cellular processes

Dynamic cellular processes often employ protein phosphorylation for rapid information transfer within and between cells. Phosphorylation-inducing chimeric small molecules (PHICS) have been developed for targeted protein phosphorylation by on-demand inducing a kinase-protein pairing. However, widespread application of PHICS has been limited as previously reported PHICS that recruited AMP-activated protein kinase (AMPK) required serum starvation and target protein overexpression, recruited only a few of the potential AMPK complexes, and exhibited poor dose- and temporal control. Herein, we report an AMPK PHICS platform that operates under physiological conditions (i.e., no serum starvation or target overexpression), recruits multiple AMPK complexes, and can induce target protein phosphorylation with dose and temporal control. We demonstrated the utility of this platform for controlling phosphorylations that underlie two dynamic cellular processes, namely oncogenic signaling and phase separation. An AMPK PHICS directed against Brutons Tyrosine Kinase (BTK), which is a driver of several B cell malignancies, was effective at inducing the death of drug-resistant cancer cells. Here, PHICS induced inhibitory phosphorylations on BTK and attenuated its oncogenic pathway. Phosphorylation of Liprin-3 induces phase separation in neurons and is critical for neurotransmitter release. Using AMPK PHICS that phosphorylated Liprin-3, we induced Liprin-3 phase separation with dose and temporal control. We envision this PHICS platform to find utility in inducing and controlling protein phosphorylations for basic research and biomedicine. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/652895v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@16bbb20org.highwire.dtl.DTLVardef@1ca5267org.highwire.dtl.DTLVardef@1e805d5org.highwire.dtl.DTLVardef@1d6ec22_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

Ultrasmall chemogenetic tags with group-transfer ligands

Chemogenetic tags are valuable tools for studying functions of a given protein-of-interest (POI) lacking small-molecule ligands, but most tags are too large for several POIs. Here, we report two ultrasmall chemogenetic tags (mgTag and cTag) of 36 and 50 amino acids (aa) that, to the best of our knowledge, are the smallest reported. These tags exhibit transferase-type reactivity with their ligands to append any moiety of interest to the tag. cTag utilizes an engineered C1 domain-bearing cysteine that undergoes group-transfer reaction with its ligand. Likewise, mgTag utilizes an engineered zinc-finger domain-bearing cysteine that undergoes group-transfer reaction with its molecular-glue ligand in the presence of cereblon (CRBN). We applied these tags in the context of cell signaling and proximity induction. While the fusion of HaloTag (297 aa) to the KRASG12D (188 aa) disrupted its ability to activate the growth-signaling pathway, fusion of mgTag or cTag did not. Group-transfer of BRD4 binder to tags appended to Abelson kinase (ABL) induced proximity between ABL and BRD4, resulting in the latters phosphorylation. Deletion of the transferase-type reactivity reduced phosphorylation levels, suggesting that proximity-inducing chimeras with group-transfer design may be more efficacious in certain scenarios. We envision these ultrasmall tags to have wide-ranging applications, including in basic science, biotechnology, and medicine. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/653252v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1f99888org.highwire.dtl.DTLVardef@18d3e0corg.highwire.dtl.DTLVardef@1e4a044org.highwire.dtl.DTLVardef@d610ee_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

Phospholipid Scramblases TMEM16F and Xkr8 regulate distinct features of Phosphatidylserine (PS) externalization and immune regulation in the tumor microenvironment to regulate tumor growth

The phospholipid scramblases Xkr8 and TMEM16F externalize phosphatidylserine (PS) by distinct mechanisms. Xkr8, is activated by caspase-mediated proteolytic cleavage, and in synergy with inactivation of P4-ATPase flippases, results in the irreversible externalization of PS on apoptotic cells and an "eat-me" signal for efferocytosis. In contrast, TMEM16F is a calcium activated scramblase that reversibly externalizes PS on viable cells via the transient increase in intracellular calcium in live cells. The tumor microenvironment (TME) is abundant with exposed PS, resulting from prolonged oncogenic and metabolic stresses and high apoptotic indexes of tumors. Such chronic PS externalization in the TME has been linked to host immune evasion from interactions of PS with inhibitory PS receptors such as TAM and TIM receptors. Here, in an effort to better understand the contributions of apoptotic vs live cell PS-externalization to tumorigenesis and immune evasion, we employed an E0771 orthotopic breast cancer model and genetically ablated Xkr8 and TMEM16F using CRISPR/Cas9. While neither the knockout of Xkr8 nor TMEM16F showed defects in cell intrinsic properties related to proliferation, tumor-sphere formation, and growth factor signaling, both knockouts suppressed tumorigenicity in immune-competent mice, but not in NOD/SCID or RAG-KO immune-deficient strains. Mechanistically, Xkr8-KO tumors suppressed macrophage-mediated efferocytosis, and TMEM16F-KO suppressed ER stress/calcium-induced PS externalization. Our data support an emerging idea in immune-oncology that constitutive PS externalization, mediated by scramblase dysregulation on tumor cells, supports immune evasion in the tumor microenvironment. This links apoptosis/efferocytosis and oncogenic stress involving calcium dysregulation, contributing to PS-mediated immune escape and cancer progression.

cancer biology↗

Small Molecule Modulators of TOX protein to Re-invigorate T cell Activity

The TOX protein (thymocyte selection-associated high mobility group box) is a critical transcription factor implicated in both T acute lymphoblastic leukemia (T-ALL) and CD8+ T cell exhaustion. Gene perturbation studies suggest that inhibiting TOX may have therapeutic implications for both leukemia and T cell exhaustion. However, due to its complex molecular mechanisms and intrinsically disordered structure, TOX has not been effectively targeted by small molecules to date. In this study, we used small molecule microarray (SMM) screening and biochemical assays to identify a series of TOX protein-protein interaction (PPI) inhibitors. We identified KI-TOX-A3 as a TOX protein binder and potent TOX PPI inhibitor. In T-ALL, KI-TOX-A3 revealed selective cytotoxicity and proteosome-dependent TOX degradation. In CD8+ T cells, KI-TOX-A3 potently reversed T cell exhaustion by decreasing surface inhibitory receptors, increasing expression of effector cytokines, and enhancing cancer cell killing activity. We also demonstrate the utility of KI-TOX-A3 to probe potential epigenetic regulatory mechanisms of TOX via KAT7 acetylation in T cells.

biochemistry↗

Protective antigen-mediated delivery of an anti-CRISPR proteinfor precision genome editing

Precise control over the dosage of Cas9-based technologies is essential because off-target effects, mosaicism, chromosomal aberrations, immunogenicity, and genotoxicity can arise with prolonged Cas9 activity. Type II anti-CRISPR proteins (Acrs) inhibit and control Cas9 but are generally impermeable to the cell membrane due to their size (6-54 kDa) and anionic charge. Moreover, existing Acr delivery methods are long-lived and operate within hours (e.g., viral and non-viral vectors) or are not applicable in vivo (e.g., nucleofection), limiting therapeutic applications. To address these problems, we developed the first protein-based anti-CRISPR delivery platform, LFN-Acr/PA, which delivers Acrs into cells within minutes. LFN-Acr/PA is a nontoxic, two-component protein system derived from anthrax toxin, where protective antigen proteins bind receptors widespread in human cells, forming a pH-triggered endosomal pore that LFN-Acr binds and uses to enter the cell. In the presence of PA, LFN-Acr enters human cells at concentrations as low as 2.5 pM to inhibit up to 95% of Cas9-mediated knockout, knock-in, transcriptional activation, and base editing. Timing LFN-Acr delivery reduces off-target base editing and increases Cas9 specificity by 41%. LFN-Acr/PA is the most potent known cell-permeable CRISPR-Cas inhibition system, significantly improving the utility of CRISPR for genome editing.

biochemistry↗