Biology subjects

Nabet, B.

Publications and source records attributed to Nabet, B..

14 recordsLinked to original sources

bioRxiv2026

De novo design of selective kinase modulators

Protein kinases are critical regulators of cellular signaling, but precise modulation of their activity remains challenging due to their high structural conservation. Here, we present de novo designed genetically encoded miniproteins capable of activating or inhibiting focal adhesion kinase (FAK) by directly targeting the kinase domain itself. Among 96 binders designed to stabilize distinct conformational states of FAK, 33 modulated kinase activity. Biochemical characterization of the four most potent modulators revealed that two designs inhibit FAK with low-nanomolar IC50 values while the remaining two potentiated FAK activity by more than two-fold. When expressed in cells, the modulators preserved the same inhibitory and activating effects observed in vitro, establishing that designed conformational binders can directly tune FAK signaling in living cells. Taking advantage of the high similarity between kinases, we redesigned the FAK inhibitors to inhibit Src kinase. Our approach establishes a versatile platform for selective and genetically encoded kinase control as a way to rewire cell signaling and as a starting point for the discovery of novel modulatory sites of kinases.

biochemistry↗

bioRxiv2026

Reversible in vivo regulation of drug metabolizing enzyme CYP1A2 activity through a dTAG knock-in strategy

Drug-metabolizing enzymes determine therapeutic exposure, efficacy and toxicity, but defining their isoform-specific functions in vivo remains challenging. Cytochrome P450 enzymes (P450s) are central to drug metabolism and pharmacokinetics (DMPK) and mediate the phase I metabolism of [~]75% of all marketed drugs. However, conventional knockout models can induce develop-mental and compensatory adaptations, and selective inhibitors are unavailable for many P450 isoforms. Here, we report the use of an inducible chemical-genetic platform for acute and specific degradation of the endogenous P450 enzyme Cyp1a2 in mice. Using CRISPR-Cas9-mediated knock-in editing, we introduced an FKBP12F36V degron into the endogenous Cyp1a2 locus to generate Cyp1a2dTAG mice. Treatment with the dTAG degrader dTAG-13 recruited an E3 ubiquitin ligase to CYP1A2dTAG, resulting in rapid and reversible proteasomal depletion of CYP1A2dTAG in vivo. Temporally controlled CYP1A2dTAG loss altered caffeine pharmacokinetics as expected, validating this model as a functional tool for DMPK studies. By enabling reversible suppression of drug-metabolizing enzymes without permanent deletion or chronic inhibitor exposure, this work establishes targeted protein degradation as a broadly adaptable strategy for studying drug metabolism in vivo and provides a foundation for extending inducible DMPK control to other P450s, conjugating enzymes and transporters.

pharmacology and toxicology↗

bioRxiv2025

Armoring STEAP1 CAR T cells with IL-18 potentiates antitumor activity in Ewing sarcoma

BackgroundEwing sarcoma (EwS) is a highly aggressive cancer driven by the EWS::FLI1 fusion oncoprotein affecting children, adolescents, and young adults. Six transmembrane epithelial antigen 1 (STEAP1) is a cell surface antigen transcriptionally controlled by EWS::FLI1 that is broadly expressed in EwS, positioning it as a rational immunotherapy target. However, translating CAR T therapy to solid tumors requires overcoming barriers to potency while maintaining safety. MethodsAnalyses of transcriptome and proteome data were performed to evaluate the effects of EWS::FLI1 perturbation on STEAP1 expression at the transcript and protein levels in EwS models. STEAP1 expression was validated in EwS patient tissues by immunohistochemistry. Second-generation STEAP1-BB{zeta} CAR T cells were tested in orthotopic and disseminated EwS xenograft models. To enhance antitumor activity, an IL-18-armored STEAP1 CAR was engineered. Dose-dependent therapeutic efficacy and safety were evaluated through measurement of tumor burden, survival, and observation for gross toxicities. ResultsSTEAP1 was expressed in [~]97% of primary EwS tumors and directly associated with EWS::FLI1 fusion protein expression in EwS cell lines. In orthotopic EwS models, STEAP1 CAR T cells induced complete tumor regression at 5 x 106 cells. In disseminated disease models, responses were dose-dependent with no evidence of antigen loss. Notably, IL-18 armored STEAP1 CAR T cells achieved complete responses in [~]80% of mice at a reduced dose of 106 cells without overt toxicity. ConclusionsThese data establish STEAP1 as a clinically relevant and highly expressed target in EwS and demonstrate that IL-18 armoring significantly improves CAR T cell efficacy by enhancing potency evident through antitumor activity at reduced cell dose. STEAP1 CAR T cells are currently under evaluation in a first-in-human phase 1/2 dose-escalation clinical trial for metastatic castration-resistant prostate cancer (NCT06236139) and these studies support future clinical translation of STEAP1 CAR T cell therapy for relapsed/refractory EwS.

cancer biology↗

bioRxiv2025

Development of PROTACs for targeted degradation of oncogenic TRK fusions

Chromosomal translocations leading to the fusion of tropomyosin receptor kinases (TRK) with diverse partner proteins have been identified as oncogenic drivers in many adult and pediatric cancers. While first-generation TRK kinase inhibitors, such as entrectinib and larotrectinib, have shown positive responses in TRK fusion-positive cancers, resistance mutations against these inhibitors in the kinase domain limit their efficacy. Second-generation inhibitors are in clinical evaluation, highlighting a need for novel therapeutic modalities to achieve durable suppression of the oncogenic activity of TRK fusions. Here, we developed heterobifunctional small molecule degraders (PROTACs) to achieve targeted degradation of TRK fusions. By conjugating entrectinib to thalidomide, we identified JWJ-01-378 as a potent and selective CRBN-recruiting degrader of the TPM3-TRKA fusion. JWJ-01-378 induced TPM3-TRKA degradation through the ubiquitin-proteasome system and proteomics analysis confirmed the acute selectivity of JWJ-01-378 for achieving TPM3-TRKA degradation with minimal off-target effects. While JWJ-01-378 was also able to degrade wild-type TRK, it was unable to degrade TRK inhibitor resistant mutants and ALK fusions. Importantly, TPM3-TRKA degradation by JWJ-01-378 suppressed downstream signaling and reduced cancer cell viability, with improved responses compared to heterobifunctional control compounds that cannot degrade TPM3-TRKA. Together, our study expands the toolbox of compounds for evaluating targeted degradation of TRK fusions in cancer. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/660465v1_ufig13.gif" ALT="Figure 13"> View larger version (19K): org.highwire.dtl.DTLVardef@6c78f7org.highwire.dtl.DTLVardef@179dcdeorg.highwire.dtl.DTLVardef@1939988org.highwire.dtl.DTLVardef@144c718_HPS_FORMAT_FIGEXP M_FIG C_FIG JWJ-01-378 recruits cereblon (CRBN) to induce potent and selective degradation of oncogenic TRK fusions, leading to a collapse in downstream signaling and loss of cancer cell viability. Graphical abstract was created using Biorender.com.

cancer biology↗

bioRxiv2024

Proteasomal control of anti-CRISPRs for the regulation of CRISPR/Cas9 activity using Cas9-ACROBAT

Small molecule-mediated proteasomal degradation of proteins is a powerful tool for synthetic regulation of biological activity. To control Cas9 activity in cells, we engineered an anti-CRISPR protein, AcrIIA4, fused to a degradation (dTAG) or small molecule assisted shutoff (SMASh) tag. Co-expression of the tagged AcrIIA4 along with Cas9 and riboswitch-regulated sgRNAs enables precise tunable control of CRISPR activity by small molecule addition.

cell biology↗

bioRxiv2023

Rapid-kinetics degron benchmarking reveals off-target activities and mixed agonism-antagonism of MYB inhibitors

Attenuating aberrant transcriptional circuits holds great promise for the treatment of numerous diseases, including cancer. However, development of transcriptional inhibitors is hampered by the lack of a generally accepted functional cellular readout to characterize their target specificity and on-target activity. We benchmarked the direct gene-regulatory signatures of six agents reported as inhibitors of the oncogenic transcription factor MYB against targeted MYB degradation in a nascent transcriptomics assay. The inhibitors demonstrated partial specificity for MYB target genes but displayed significant off-target activity. Unexpectedly, the inhibitors displayed bimodal on-target effects, acting as mixed agonists-antagonists. Our data uncover unforeseen agonist effects of small molecules originally developed as TF inhibitors and argue that rapid-kinetics benchmarking against degron models should be used for functional characterization of transcriptional modulators.

molecular biology↗

bioRxiv2023

Automated tumor immunophenotyping predicts clinical benefit from anti-PD-L1 immunotherapy

BackgroundCancer immunotherapy has transformed the clinical approach to patients with malignancies as profound benefits can be seen in a subset of patients. To identify this subset, biomarker analyses increasingly focus on phenotypic and functional evaluation of the tumor microenvironment (TME) to determine if density, spatial distribution, and cellular composition of immune cell infiltrates can provide prognostic and/or predictive information. Attempts have been made to develop standardized methods to evaluate immune infiltrates in the routine assessment of certain tumor types; however, broad adoption of this approach in clinical decision-making is still missing. MethodsWe developed approaches to categorize solid tumors into "Desert", "Excluded" and "Inflamed" types according to the spatial distribution of CD8+ immune effector cells to determine the prognostic and/or predictive implications of such labels. To overcome the limitations of this subjective approach we incrementally developed four automated analysis pipelines of increasing granularity and complexity for density and pattern assessment of immune effector cells. ResultsWe show that categorization based on "manual" observation is predictive for clinical benefit from anti-programmed cell death ligand-1 (PD-L1) therapy in two large cohorts of patients with non-small cell lung cancer (NSCLC) or triple-negative breast cancer (TNBC). For the automated analysis we demonstrate that a combined approach outperforms individual pipelines and successfully relates spatial features to pathologist-based read-outs and patient response to therapy. ConclusionsOur findings suggest tumor immunophenotype (IP) generated by automated analysis pipelines should be evaluated further as potential predictive biomarkers for cancer immunotherapy. What is already known on this topicClinical benefit from checkpoint inhibitor-targeted therapies is realized only in a subset of patients. Robust biomarkers to identify patients who may respond to such therapies are needed. What this study addsWe have developed manual and automated approaches to categorize tumors into immunophenotypes based on the spatial distribution of CD8+ T effector cells that predict clinical benefit from anti-PD-L1 immunotherapy for patients with advanced non-small cell lung cancer or triple-negative breast cancer. How this study might affect research, practice or policyTumor immunophenotypes should be further validated as predictive biomarker for checkpoint inhibitor-targeted therapies in prospective clinical studies.

pathology↗

bioRxiv2023

Leukemia core transcriptional circuitry is a sparsely interconnected hierarchy stabilized by incoherent feed-forward loops

Lineage-defining transcription factors form densely interconnected circuits in chromatin occupancy assays, but the functional significance of these networks remains underexplored. We reconstructed the functional topology of a leukemia cell transcription network from the direct gene-regulatory programs of eight core transcriptional regulators established in pre-steady state assays coupling targeted protein degradation with nascent transcriptomics. The core regulators displayed narrow, largely non-overlapping direct transcriptional programs, forming a sparsely interconnected functional hierarchy stabilized by incoherent feed-forward loops. BET bromodomain and CDK7 inhibitors disrupted the core regulators direct programs, acting as mixed agonists/antagonists. The network is predictive of dynamic gene expression behaviors in time-resolved assays and clinically relevant pathway activity in patient populations.

systems biology↗

bioRxiv2022

Chemical degradation of BTK/TEC as a novel approach to inhibit platelet function and thrombosis.

The tyrosine kinase BTK plays an important role in platelet function downstream of GPVI and CLEC2 receptors and has been proposed as a novel target to prevent thrombosis in patients that are at increased risk. However, current clinically approved BTK inhibitors have off target effects and are associated with an increased bleeding risk. In this study, we therefore explored whether BTK can be targeted for degradation in human platelets by using recently developed heterobifunctional molecules that employ the proteasomal system to break down BTK. Here we confirm that human platelets are highly susceptible to BTK degraders with the generic tyrosine kinase degrader TL12-186, and the BTK degraders DD-04-15 and DD-03-171 leading to breakdown of BTK and its closely related kinase TEC, an effect that was prevented by proteasomal inhibitors. Tandem Mass Tag proteomic analysis confirmed high selectivity with TL12-186 degrading BTK/TEC, FAK/PYK2 and FER, whereas DD-04-15 and DD-03-171 degraded BTK/TEC only. GPVI-mediated platelet integrin IIb{beta}3 activation, P-selectin expression, and phosphatidyl-serine exposure were largely impaired upon BTK/TEC degradation, with PAR-1-mediated responses left intact. This is the first study to demonstrate that chemical protein degraders can be successfully employed in anucleate human platelets to modulate their function.

cell biology↗

bioRxiv2021

A distinct core regulatory module enforces oncogene expression in KMT2A-rearranged leukemia

A small set of lineage-restricted transcription factors (TFs), termed core regulatory circuitry (CRC), control cell identity and malignant transformation. Here, we integrated gene dependency, chromatin architecture and TF perturbation datasets to characterize 31 core TFs in acute myeloid leukemia (AML). Contrary to a widely accepted model, we detected a modular CRC structure with hierarchically organized, partially redundant and only sparsely interconnected modules of core TFs controlling distinct genetic programs. Rapid TF degradation followed by measurement of genome-wide transcription rates revealed that core TFs directly regulate dramatically fewer genes than previously assumed. Leukemias carrying KMT2A (MLL) rearrangements depend on the IRF8/MEF2 axis to directly enforce expression of the key oncogenes MYC, HOXA9 and BCL2. Our datasets provide an evolving model of CRC organization in human cells, and a resource for further inquiries into and therapeutic targeting of aberrant transcriptional circuits in cancer.

genomics↗

bioRxiv2021

Effects of forced cohesin eviction and retention on X-inactivation and autosomes

Depletion of architectural factors globally alters chromatin structure, but only modestly affects gene expression. We revisit the structure-function relationship using the inactive X chromosome (Xi) as a model. We investigate cohesin imbalances by forcing its depletion or retention using degron-tagged RAD21 (cohesin subunit) or WAPL (cohesin release factor). Interestingly, cohesin loss disrupts Xi superstructure, unveiling superloops between escapee genes, with minimal effect on gene repression. By contrast, forced cohesin retention markedly affects Xi superstructure and compromises spreading of Xist RNA-Polycomb complexes, attenuating Xi silencing. Effects are greatest at distal chromosomal ends, where looping contacts with the Xist locus are weakened. Surprisingly, cohesin loss created an "Xi superloop" and cohesin retention created "Xi megadomains" on the active X. Across the genome, a proper cohesin balance protects against aberrant inter-chromosomal interactions and tempers Polycomb-mediated repression. We conclude that a balance of cohesin eviction and retention regulates X-inactivation and inter-chromosomal interactions across the genome.

molecular biology↗

bioRxiv2020

Mammalian Cell Proliferation Requires Noncatalytic Functions of O-GlcNAc Transferase

O-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting OGTs myriad cellular roles has been challenging because genetic deletion is lethal; complementation methods have not been established. Here, we developed approaches to replace endogenous OGT with separation-of-function variants to investigate the importance of OGTs enzymatic activities for cell viability. Using genetic complementation, we found that OGTs glycosyltransferase function is required for cell growth but its protease function is dispensable. We next used complementation to construct a cell line with degron-tagged wild-type OGT. When OGT was degraded to very low levels, cells stopped proliferating but remained viable. Adding back catalytically-inactive OGT rescued growth. Therefore, OGT has an essential noncatalytic role that is necessary for cell proliferation. By developing a method to quantify how OGTs catalytic and noncatalytic activities affect protein abundance, we found that OGTs noncatalytic functions often affect different proteins from its catalytic functions. Proteins involved in oxidative phosphorylation and the actin cytoskeleton were especially impacted by the noncatalytic functions. We conclude that OGT integrates both catalytic and noncatalytic functions to control cell physiology. SignificanceMammalian cells contain only one glycosyltransferase, OGT, that operates in the nucleus and cytoplasm rather than the secretory pathway. OGT is required for cell proliferation, but a basic unanswered question is what OGT functions are essential. This question is challenging to address because OGT has thousands of glycosylation substrates, two different enzymatic activities, and a large number of binding partners. Here, by establishing genetic tools to replace endogenous OGT with variants that preserve only a subset of its activities, we show that only a low level of glycosylation activity is required to maintain cell viability; however, cell proliferation requires noncatalytic OGT function(s). The ability to replace OGT with variants provides a path to identifying its essential substrates and binding partners.

biochemistry↗

bioRxiv2020

Sulfopin, a selective covalent inhibitor of Pin1, blocks Myc-driven tumor initiation and growth in vivo

The peptidyl-prolyl cis-trans isomerase, Pin1, acts as a unified signaling hub that is exploited in cancer to activate oncogenes and inactivate tumor suppressors, in particular through up-regulation of c-Myc target genes. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to discover covalent inhibitors targeting Pin1s active site nucleophile - Cys113, leading to the development of Sulfopin, a double-digit nanomolar Pin1 inhibitor. Sulfopin is highly selective for Pin1, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement, and phenocopies genetic knockout of Pin1. Although Pin1 inhibition had a modest effect on viability in cancer cell cultures, Sulfopin induced downregulation of c-Myc target genes and reduced tumor initiation and tumor progression in murine and zebrafish models of MYCN-driven neuroblastoma. Our results suggest that Sulfopin is a suitable chemical probe for assessing Pin1-dependent pharmacology in cells and in vivo. Moreover, these studies indicate that Pin1 should be further investigated as a potential cancer target.

cancer biology↗

bioRxiv2020

Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

Chemical biology strategies for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAGV-1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12F36V-tagged proteins. dTAGV-1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, supports combination degrader studies and facilitates investigations of protein function in cells and mice.

cancer biology↗