RNA Labeling Research Articles

Subcellular mRNA kinetic modeling reveals nuclear retention as rate-limiting.

Eukaryotic mRNAs are transcribed, processed, translated, and degraded in different subcellular compartments. Here, we measured mRNA flow rates between subcellular compartments in mouse embryonic stem cells. By combining metabolic RNA labeling, biochemical fractionation, mRNA sequencing, and mathematical modeling, we determined the half-lives of nuclear pre-, nuclear mature, cytosolic, and membrane-associated mRNAs from over 9000 genes. In addition, we estimated transcript elongation rates. Many matured mRNAs have long nuclear half-lives, indicating nuclear retention as the rate-limiting step in the flow of mRNAs. In contrast, mRNA transcripts coding for transcription factors show fast kinetic rates, and in particular short nuclear half-lives. Differentially localized mRNAs have distinct rate constant combinations, implying modular regulation. Membrane stability is high for membrane-localized mRNA and cytosolic stability is high for cytosol-localized mRNA. mRNAs encoding target signals for membranes have low cytosolic and high membrane half-lives with minor differences between signals. Transcripts of nuclear-encoded mitochondrial proteins have long nuclear retention and cytoplasmic kinetics that do not reflect co-translational targeting. Our data and analyses provide a useful resource to study spatiotemporal gene expression regulation.

Nascent and Mature RNA Profiling by Subcellular Fractionation in Human Cells.

Transcription and RNA decay determine steady-state RNA levels in cells available for translation and RNA-mediated regulatory functions. Both processes can be assessed by various techniques, for majority, based on RNA labelling or chromatin immunoprecipitation, but require a high level of expertise. Here, we describe a cost-effective, fast, and simple protocol that enables the profiling of nascent and mature RNA in the cytoplasm, nucleoplasm, and chromatin through subcellular fractionation. The workflow can include α-amanitin inhibition of RNA Polymerase II to assess nascent RNAs as a proxy of transcriptional activity, or it can be used without this treatment to investigate distribution of partially processed or mature transcripts across distinct subcellular compartments. It is applicable for studying any of RNA biotypes, including small and long noncoding RNAs, mRNAs, and their splice variants, on both transcript-specific and transcriptome-wide scales. Nascent or mature RNAs isolated from each fraction can be further analyzed by any technique of choice (northern blot, reverse transcription, RNA sequencing).

Imaging of Viral Genomic RNA Replication with Nanoprobes.

Viruses are a great threat to human life and health. Different viruses have its unique mechanism to efficiently infect cells, and the entry process and the nucleic acid replication using cell machine are two critical processes related to the fate of virus progeny. Real-time and long-term imaging techniques can be used to thoroughly investigate the viral infection events. This chapter will present the labeling of viral genomic RNA (gRNA) replication by developing new nanoprobes, one-donor-two-acceptors one, for example, in which the synergistic coupling of multiple energy transfer strategy, so as that the journey of viruses in live cells could be monitored and imaged in real time. Methods of labeling as well as that used for fluorescent and dark field scattering imaging are outlined.

SUPREM: an engineered non-site-specific m6A RNA methyltransferase with highly improved efficiency.

N 6-Methyladenine(m6A) RNA methylation plays a key role in RNA processing and translational regulation, influencing both normal physiological and pathological processes. Yet, current techniques for studying RNA methylation struggle to isolate the effects of individual m6A modifications. Engineering of RNA methyltransferases (RNA MTases) could enable development of improved synthetic biology tools to manipulate RNA methylation, but it is challenging due to limited understanding of structure-function relationships in RNA MTases. Herein, using ancestral sequence reconstruction, we explore the sequence space of the bacterial DNA methyltransferase EcoGII (M.EcoGII), a promising target for protein engineering due to its lack of sequence specificity and its residual activity on RNA. We thereby created an efficient non-specific RNA MTase termed SUPer RNA EcoGII Methyltransferase (SUPREM), which exhibits 8-fold higher expression levels, 7°C higher thermostability and 12-fold greater m6A RNA methylation activity compared with M.EcoGII. Immunofluorescent staining and quantitativeliquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed SUPREM's higher RNA methylation activity compared with M.EcoGII in mammalian cells. Additionally, Nanopore direct RNA sequencing highlighted that SUPREM is capable of methylating a larger number of RNA methylation sites than M.EcoGII. Through phylogenetic and mutational analysis, we identified a critical residue for the enhanced RNA methylation activity of SUPREM. Collectively, our findings indicate that SUPREM holds promise as a versatile tool for in vivo RNA methylation and labeling.

APEX2 proximity labeling of RNA in bacteria.

Studies over the past several years have shown that distinct RNAs can be targeted to subcellular locations in bacterial cells. The ability to investigate localized RNAs in bacteria is currently limited to imaging-based approaches or to laborious procedures to isolate ribonucleoprotein complexes by grad-seq, HITS-CLIP, or Rloc-seq. However, a major challenge in studying mRNA localization in bacterial cells is that bacterial mRNAs typically last for only a few minutes in the cell, while experiments to investigate their localization or interaction partners can take much longer. Therefore, rapid methods of studying RNA localization are needed to bridge this technical challenge.

Metabolic RNA labeling in non-engineered cells following spontaneous uptake of fluorescent nucleoside phosphate analogues.

RNA and its building blocks play central roles in biology and have become increasingly important as therapeutic agents and targets. Hence, probing and understanding their dynamics in cells is important. Fluorescence microscopy offers live-cell spatiotemporal monitoring but requires labels. We present two fluorescent adenine analogue nucleoside phosphates which show spontaneous uptake and accumulation in cultured human cells, likely via nucleoside transporters, and show their potential utilization as cellular RNA labels. Upon uptake, one nucleotide analogue, 2CNqAXP, localizes to the cytosol and the nucleus. We show that it could then be incorporated into de novo synthesized cellular RNA, i.e.it was possible to achieve metabolic fluorescence RNA labeling without using genetic engineering to enhance incorporation, uptake-promoting strategies, or post-labeling through bio-orthogonal chemistries. By contrast, another nucleotide analogue, pAXP, only accumulated outside of the nucleus and was rapidly excreted. Consequently, this analogue did not incorporate into RNA. This difference in subcellular accumulation and retention results from a minor change in nucleobase chemical structure. This demonstrates the importance of careful design of nucleoside-based drugs, e.g. antivirals to direct their subcellular localization, and shows the potential of fine-tuning fluorescent base analogue structures to enhance the understanding of the function of such drugs.

High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues

RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts.

Time-resolved scRNA-seq reveals transcription dynamics of polarized macrophages with influenza A virus infection and antigen presentation to T cells

Throughout history, the influenza A virus has caused numerous devastating global pandemics. Macrophages, as pivotal innate immune cells, exhibit a wide range of immune functions characterized by distinct polarization states, reflecting their intricate heterogeneity. In this study, we employed the time-resolved single-cell sequencing technique coupled with metabolic RNA labelling to elucidate the dynamic transcriptional changes in distinct polarized states of bone marrow-derived macrophages (BMDMs) upon infection with the influenza A virus. Our approach not only captures the temporal dimension of transcriptional activity, which is lacking in conventional scRNA-seq methods, but also reveals that M2-polarized Arg1_macrophages is the sole state supporting successful replication of influenza A virus. Furthermore, we identified distinct antigen presentation capabilities to CD4 + T and CD8 + T cells across diverse polarized states of macrophages. Notably, the M1 phenotype, exhibited by both bone marrow-derived macrophages (BMDMs) and murine alveolar macrophages (AMs), demonstrated superior conventional and cross-presentation abilities for exogenous antigens, with a particular emphasis on cross-presentation capacity. Additionally, as CD8 + T cell differentiation progressed, M1 polarization exhibited an enhanced capacity for cross-presentation. All three phenotypes of BMDMs, including M1, demonstrated robust presentation of CD4 + regulatory T cells, while displaying limited ability to present naive CD4+ T cells. These findings offer novel insights into the immunological regulatory mechanisms governing distinct polarized states of macrophages, particularly their roles in restricting the replication of influenza A virus and modulating antigen-specific T cell responses through innate immunity.

Expanding the Available RNA Labeling Toolbox With CutA Nucleotidyltransferase for Efficient Transcript Labeling with Purine and Pyrimidine Nucleotide Analogs.

RNA labeling is an invaluable tool for investigation of the function and localization of nucleic acids. Labels are commonly incorporated into 3' end of RNA and the primary enzyme used for this purpose is RNA poly(A) polymerase (PAP), which belongs to the class of terminal nucleotidyltransferases (NTases). However, PAP preferentially adds ATP analogs, thus limiting the number of available substrates. Here, we report the use of another NTase, CutA from the fungus Thielavia terrestris. Using this enzyme, we were able to incorporate into the 3' end of RNA not only purine analogs, but also pyrimidine analogs. We engaged strain-promoted azide-alkyl cycloaddition (SPAAC) to obtain fluorescently labeled or biotinylated transcripts from RNAs extended with azide analogs by CutA. Importantly, modified transcripts retained their biological properties. Furthermore, fluorescently labeled mRNAs were suitable for visualization in cultured mammalian cells. Finally, we demonstrate that either affinity studies or molecular dynamic (MD) simulations allow for rapid screening of NTase substrates, what opens up new avenues in the search for the optimal substrates for this class of enzymes.

Phase Separation Modulates the Thermodynamics and Kinetics of RNA Hybridization.

Biomolecular condensates can influence cellular function in a number of ways, including by changing the structural dynamics and conformational equilibria of the molecules partitioned within them. Here we use methyl transverse relaxation optimized spectroscopy (methyl-TROSY) NMR in conjunction with 2'-O-methyl labeling of RNA to characterize the thermodynamics and kinetics of RNA-RNA base pairing in condensates formed by the C-terminal intrinsically disordered region of CAPRIN1, an RNA-binding protein involved in RNA transport, translation, and stability. CAPRIN1 condensates destabilize RNA-RNA base pairing, resulting from a ∼270-fold decrease and a concomitant ∼15-fold increase in the on- and off-rates for duplex formation, respectively. The ∼30-fold slower diffusion of RNA single strands within the condensed phase partially accounts for the reduced on-rate, but the further ∼9-fold reduction likely reflects shedding of CAPRIN1 chains that are interacting with the RNA prior to hybridization. Our study emphasizes the important role of protein solvation in modulating nucleic acid recognition processes inside condensates.

Biocatalytic Nucleobase Diversification of 4'-Thionucleosides and Application of Derived 5-Ethynyl-4'-thiouridine for RNA Synthesis Detection.

Nucleoside and nucleotide analogues have proven to be transformative in the treatment of viral infections and cancer. One branch of structural modification to deliver new nucleoside analogue classes explores replacement of canonical ribose oxygen with a sulfur atom. Whilst biological activity of such analogues has been shown in some cases, widespread exploration of this compound class is hitherto hampered by the lack of a straightforward and universal nucleobase diversification strategy. Herein, we present a synergistic platform enabling both biocatalytic nucleobase diversification from 4'-thiouridine in a one-pot process, and chemical functionalization to access new entities. This methodology delivers entry across pyrimidine and purine 4'-thionucleosides, paving a way for wider synthetic and biological exploration. We exemplify our approach by enzymatic synthesis of 5-iodo-4'-thiouridine on multi-milligram scale and from here switch to complete chemical synthesis of a novel nucleoside analogue probe, 5-ethynyl-4'-thiouridine. Finally, we demonstrate the utility of this probe to monitor RNA synthesis in proliferating HeLa cells, validating its capability as a new metabolic RNA labelling tool.

Biocatalytic Nucleobase Diversification of 4′‐Thionucleosides and Application of Derived 5‐Ethynyl‐4′‐thiouridine for RNA Synthesis Detection

AbstractNucleoside and nucleotide analogues have proven to be transformative in the treatment of viral infections and cancer. One branch of structural modification to deliver new nucleoside analogue classes explores replacement of canonical ribose oxygen with a sulfur atom. Whilst biological activity of such analogues has been shown in some cases, widespread exploration of this compound class is hitherto hampered by the lack of a straightforward and universal nucleobase diversification strategy. Herein, we present a synergistic platform enabling both biocatalytic nucleobase diversification from 4′‐thiouridine in a one‐pot process, and chemical functionalization to access new entities. This methodology delivers entry across pyrimidine and purine 4′‐thionucleosides, paving a way for wider synthetic and biological exploration. We exemplify our approach by enzymatic synthesis of 5‐iodo‐4′‐thiouridine on multi‐milligram scale and from here switch to complete chemical synthesis of a novel nucleoside analogue probe, 5‐ethynyl‐4′‐thiouridine. Finally, we demonstrate the utility of this probe to monitor RNA synthesis in proliferating HeLa cells, validating its capability as a new metabolic RNA labelling tool.

Splice_sim: a nucleotide conversion-enabled RNA-seq simulation and evaluation framework

Nucleotide conversion RNA sequencing techniques interrogate chemical RNA modifications in cellular transcripts, resulting in mismatch-containing reads. Biases in mapping the resulting reads to reference genomes remain poorly understood. We present splice_sim, a splice-aware RNA-seq simulation and evaluation pipeline that introduces user-defined nucleotide conversions at set frequencies, creates mixture models of converted and unconverted reads, and calculates mapping accuracies per genomic annotation. By simulating nucleotide conversion RNA-seq datasets under realistic experimental conditions, including metabolic RNA labeling and RNA bisulfite sequencing, we measure mapping accuracies of state-of-the-art spliced-read mappers for mouse and human transcripts and derive strategies to prevent biases in the data interpretation.

Nuclear export is a limiting factor in eukaryotic mRNA metabolism.

The eukaryotic mRNA life cycle includes transcription, nuclear mRNA export and degradation. To quantify all these processes simultaneously, we perform thiol-linked alkylation after metabolic labeling of RNA with 4-thiouridine (4sU), followed by sequencing of RNA (SLAM-seq) in the nuclear and cytosolic compartments of human cancer cells. We develop a model that reliably quantifies mRNA-specific synthesis, nuclear export, and nuclear and cytosolic degradation rates on a genome-wide scale. We find that nuclear degradation of polyadenylated mRNA is negligible and nuclear mRNA export is slow, while cytosolic mRNA degradation is comparatively fast. Consequently, an mRNA molecule generally spends most of its life in the nucleus. We also observe large differences in the nuclear export rates of different 3'UTR transcript isoforms. Furthermore, we identify genes whose expression is abruptly induced upon metabolic labeling. These transcripts are exported substantially faster than average mRNAs, suggesting the existence of alternative export pathways. Our results highlight nuclear mRNA export as a limiting factor in mRNA metabolism and gene regulation.

Key progresses of MOE key laboratory of macromolecular synthesis and functionalization in 2023

In 2023, The MOE Key Laboratory of Macromolecular Synthesis and Functionalization in Zhejiang University had achieved several important results in the five research directions. First, for controllable catalytic polymerization, a new silicon-centered organoboron binary catalyst was developed for copolymerization of epoxides, and a series of cooperative organocatalysts were proposed for ring-opening copolymerization of chalcogen-rich monomers. Second, with respect to microstructure and rheology, axially encoded metafiber demonstrated its capacity for integrating multiple electronics, while artificial nacre materials showed improved strength and toughness due to interlayer entanglement. Third, concerning separating functional polymers, interfacial polymerization was monitored via aggregation-induced emission, and vacuum filtration was applied to assist interfacial polymerization. Fourth, in terms of biomedical functional polymers, we designed antibacterial materials such as a novel quaternary ammonium salt that enables polyethylene terephthalate recycling and its antibacterial function, nanozyme-armed phage proved its efficiency in combating bacterial infection, and also transition metal nanoparticles showed capacities in antibacterial treatments. We also made achievements in biomedical materials, including polymeric microneedles for minimally invasive implantation and functionalization of cardiac patches, as well as ROS-responsive/scavenging prodrug/miRNA balloon coating to promote drug delivery efficiency. Besides, methods and mechanisms of RNA labeling has been developed. Fifth, about photo-electro-magnetic functional polymers, through-space conjugation was successfully manipulated by altering subunit packing modes, room-temperature phosphorescent hydrogels were synthesized via polymerization-induced crystallization of dopant molecules, and single crystals of both fullerene and non-fullerene acceptors were grown in crystallized organogel, with their photodetection performance further explored. The related works are reviewed in this paper.

Masked acid chlorides for proximity labelling of RNA.

Masked acid chlorides for proximity labelling of RNA.

Bioorthogonal masked acylating agents for proximity-dependent RNA labelling.

RNA localization is highly regulated, with subcellular organization driving context-dependent cell physiology. Although proximity-based labelling technologies that use highly reactive radicals or carbenes provide a powerful method for unbiased mapping of protein organization within a cell, methods for unbiased RNA mapping are scarce and comparably less robust. Here we develop α-alkoxy thioenol and chloroenol esters that function as potent acylating agents upon controlled ester unmasking. We pair these probes with subcellular-localized expression of a bioorthogonal esterase to establish a platform for spatial analysis of RNA: bioorthogonal acylating agents for proximity labelling and sequencing (BAP-seq). We demonstrate that, by selectively unmasking the enol probe in a locale of interest, we can map RNA distribution in membrane-bound and membrane-less organelles. The controlled-release acylating agent chemistry and corresponding BAP-seq method expand the scope of proximity labelling technologies and provide a powerful approach to interrogate the cellular organization of RNAs.

NMR characterization and ligand binding site of the stem-loop 2 motif from the Delta variant of SARS-CoV-2.

The stem-loop 2 motif (s2m) in SARS-CoV-2 (SCoV-2) is located in the 3'-UTR. Although s2m has been reported to display characteristics of a mobile genomic element that might lead to an evolutionary advantage, its function has remained unknown. The secondary structure of the original SCoV-2 RNA sequence (Wuhan-Hu-1) was determined by NMR in late 2020, delineating the base-pairing pattern and revealing substantial differences in secondary structure compared to SARS-CoV-1 (SCoV-1). The existence of a single G29742-A29756 mismatch in the upper stem of s2m leads to its destabilization and impedes a complete NMR analysis. With Delta, a variant of concern has evolved with one mutation compared to the original sequence that replaces G29742 by U29742. We show here that this mutation results in a more defined structure at ambient temperature accompanied by a rise in melting temperature. Consequently, we were able to identify >90% of the relevant NMR resonances using a combination of selective RNA labeling and filtered 2D NOESY as well as 4D NMR experiments. We present a comprehensive NMR analysis of the secondary structure, (sub)nanosecond dynamics, and ribose conformation of s2m Delta based on heteronuclear 13C NOE and T 1 measurements and ribose carbon chemical shift-derived canonical coordinates. We further show that the G29742U mutation in Delta has no influence on the druggability of s2m compared to the Wuhan-Hu-1 sequence. With the assignment at hand, we identify the flexible regions of s2m as the primary site for small molecule binding.

203 RNA Polymerase II Meningioma Mutations Activate Forebrain Neurodevelopmental Pathways

INTRODUCTION: RNA polymerase II, which transcribes DNA into RNA, is mutated in a subset of skull base meningiomas. Patient-derived Pol II mutant meningiomas are dysregulated in pathways directing neural crest differentiation and neurodevelopment. The molecular mechanism of the Pol II driver mutation has remained elusive, and may provide novel therapeutic insight. METHODS: The POLR2A Q403K meningioma driver mutation was modeled in the mouse embryonic stem cell system using CRISPR-Cas9. Metabolic labeling of nascent RNA was performed with 4-thiouridine (SLAMseq). Pol II ChIPseq was performed to calculate the travel ratio (proportion of initiated Pol II to elongating Pol II). POLR2A Q403K and wildtype mES cells differentiated over 12 days in permissive media, RNA was harvested over multiple days, and RNAseq was performed. RESULTS: POLR2A Q403K cells had a global increase in transcription per nascent RNA sequencing, as measured by T>C conversions (p = 5.71x10-66). For active and highly-paused genes, which were enriched for neurodevelopmental pathways (FDR = 3.52x10-25), a left shift in the mean travel ratio revealed defects in pause-release as the mechanism for increased levels of nascent transcription. Recapitulating human disease, POLR2A Q403K mES cells had significant activation of Hedgehog (Padj. = 2.4x10-3) and WNT (Padj. = 5.2x10-3) signaling pathways, and by day 12 of differentiation showed significant upregulation of pathways critical for rostral cortical development including forebrain neuronal differentiation (FDR = 4.86x10-4), telencephalon development (FDR = 4.30x10-4), and neuronal differentiation (FDR = 4.39x10-4). CONCLUSIONS: Meningioma Pol II mutations drive global increases in transcription via dysregulation of pause-release. Highly-paused genes, including master regulators of neural development, are disproportionately activated by mutant Pol II. This dysregulation leads to aberrant induction of neurodevelopmental signaling, including Hedgehog and WNT, and provides a rationale for pharmacologic targeting of these pathways.

Abstract 2067: QUANTROseq, a transcriptomic based drug discovery platform using time-resolved gene expression profiling: Identification of inhibitors and degraders of transcription factors and cell signaling regulators

Abstract Integrative analyses of CRISPR/Cas9 screening datasets from different sources revealed - besides known oncogenes - a series of transcriptional master regulators as most prominent context-specific dependencies in cancer that remained largely inaccessible to conventional drug discovery approaches. New chemical entities (NCEs) that specifically interfere with a transcription factor (TF) at any level should induce and be detectable based on selective effects on their primary biochemical activity - i.e., the transcriptional activation of specific target genes. However, the definition of direct targets of a TF and, more generally, the unbiased detection of direct transcriptional effects has remained challenging due to the limited time resolution of conventional techniques for gene perturbation and transcriptional profiling. To overcome this limitation, QUANTRO Therapeutics relies on SLAMseq technology that, by metabolic labeling de novo-synthesized RNA immediately after intervention and conserving the information for later readout, provides unprecedent time-resolution in transcriptomic analysis. Aiming to target previously undruggable targets, we set out to first build definitive knowledge around their direct effect on transcription, inducing their quick protein degradation through the auxin-inducible degron (AID) technology followed by immediate transcriptional readout though SLAMseq -before any secondary, confounding effect could appear in the cell. The targets´ mRNA fingerprints produced are then used as a reference and cross-compared with existing and new chemical entities to: (i) benchmark the real mode of action of existing drugs; (ii) identify new drug candidates that specifically interfere with disease-causing transcriptional programs in cancer; (iii) interrogate the efficiency, selectivity, and potency of drug candidates during Hit-validation and Hit-to-Lead activities. Proof of concept data demonstrated that time-resolved transcriptional profiling can reveal drugs´ mode of action with unprecedent precision and sensitivity, when drugs act on Targets that have a direct effect on transcriptional regulation - such as TFs and signaling pathways. Building on this knowledge, QUANTRO Therapeutics unleashed the unique potential of mRNA fingerprinting technology for HTS-compatible drug discovery by (1) establishing scalable and automated workflows for metabolic RNA labeling, treatment of cells, and RNA extraction; (2) developing algorithms for combinatorial mRNA fingerprint-readout that allow for the simultaneous assessment of TF signatures by multiplexed targeted RNA sequencing (QUANTROseq), thus transforming the precision and scope of cell-based compound screens. Citation Format: Arianna Sabò, Nina Fasching Griesmayr, Tobias Neumann, Raphael Manzenreither, Maja Ivankovic, Rodrigo Pacheco Valamatos Costa, Sarah Rieser, Sara Scinicariello, Petr Triska, Anna Stingeder, Astrid Gruss, Ryan Cubero, Olga Frank, Ivica Sowemimo, Paul Kirchgatterer, Adriana Cantoran, Stefan Ameres, Johannes Zuber, Michael Bauer. QUANTROseq, a transcriptomic based drug discovery platform using time-resolved gene expression profiling: Identification of inhibitors and degraders of transcription factors and cell signaling regulators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2067.