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NPM1 is an abundant nucleolar chaperone that, in addition to facilitating ribosome biogenesis, contributes to nucleolar stress responses and tumor suppression through its regulation of the p14 Alternative Reading Frame tumor suppressor protein (p14 ARF ). Oncogenic stress induces p14 ARF to inhibit MDM2, stabilize p53 and arrest the cell cycle. Under non-stress conditions, NPM1 stabilizes p14 ARF in nucleoli, preventing its degradation and blocking p53 activation. However, the mechanisms underlying the regulation of p14 ARF by NPM1 are unclear because the structural features of the p14 ARF -NPM1 complex were elusive. Here we show that p14 ARF assembles into a gel-like meso-scale network upon phase separation with NPM1. This assembly is mediated by intermolecular contacts formed by hydrophobic residues in an α-helix and β-strands within a partially folded N-terminal portion of p14 ARF . These hydrophobic interactions promote phase separation with NPM1, enhance p14 ARF nucleolar partitioning, restrict NPM1 diffusion within condensates and nucleoli, and reduce cellular proliferation. Our structural analysis provides insights into the multifaceted chaperone function of NPM1 in nucleoli by mechanistically linking the nucleolar localization of p14 ARF to its partial folding and meso-scale assembly upon phase separation with NPM1., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

There are few options for patients with relapse/refractory B-cell acute lymphoblastic leukemia (B-ALL), thus this is a major area of unmet medical need. Here, we reveal that inclusion of a poison exon in RBM39, which could be induced both by CDK9 or CDK9 independent CMGC (cyclin-dependent kinases, mitogen-activated protein kinases, glycogen synthase kinases, CDC-like kinases) kinase inhibition, is recognized by the nonsense-mediated mRNA decay (NMD) pathway for degradation. Targeting this poison exon in RBM39 with CMGC inhibitors lead to protein downregulation and inhibition of ALL growth, particularly in relapse/refractory B-ALL. Mechanistically, disruption of co-transcriptional splicing by inhibition of CMGC kinases including DYRK1A, or inhibition of CDK9, which phosphorylate the C-terminal domain of RNA polymerase II (Pol II), results in alteration of SF3B1 and Pol II association. Disruption of SF3B1 and transcriptional elongation complex alters Pol II pausing, which promotes the inclusion of a poison exon in RBM39. Moreover, RBM39 ablation suppresses the growth of human B-ALL, and targeting RBM39 with sulfonamides, which degrade RBM39 protein, showed strong anti-tumor activity in preclinical models. Our data reveal that relapse/refractory B-ALL is susceptible to pharmacologic and genetic inhibition of RBM39 and provide two potential strategies to target this axis., (Copyright © 2024 American Society of Hematology.)

Intrinsically disordered regions (IDRs) within human proteins play critical roles in cellular information processing, including signaling, transcription, stress response, DNA repair, genome organization, and RNA processing. Here, we summarize current challenges in the field and propose cutting-edge approaches to address them in physiology and disease processes, with a focus on cancer., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The nucleolus is the largest biomolecular condensate and facilitates transcription, processing, and assembly of ribosomal RNA (rRNA). Although nucleolar function is thought to require multiphase liquid-like properties, nucleolar fluidity and its connection to the highly coordinated transport and biogenesis of ribosomal subunits are poorly understood. Here, we use quantitative imaging, mathematical modeling, and pulse-chase nucleotide labeling to examine nucleolar material properties and rRNA dynamics. The mobility of rRNA is several orders of magnitude slower than that of nucleolar proteins, with rRNA steadily moving away from the transcriptional sites in a slow (∼1 Å/s), radially directed fashion. This constrained but directional mobility, together with polymer physics-based calculations, suggests that nascent rRNA forms an entangled gel, whose constant production drives outward flow. We propose a model in which progressive maturation of nascent rRNA reduces its initial entanglement, fluidizing the nucleolar periphery to facilitate the release of assembled pre-ribosomal particles., Competing Interests: Declaration of interests C.P.B. is a founder and consultant for Nereid Therapeutics., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Although it is understood that myriad proteins function in cells within biomolecular condensates, it is unclear how protein condensation is altered in human disease. In this issue of Developmental Cell, Banani et al. show that mutations in disease-associated proteins may map to phase-separation-prone regions and thereby alter condensate formation., Competing Interests: Declaration of interests R.K. has received compensation unrelated to this work for advisory board membership of Dewpoint Therapeutics, for consulting with GLG Consulting, and through patent licensing to New Equilibrium Biosciences., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Biomolecular condensates are cellular organelles formed through liquid-liquid phase separation (LLPS) that play critical roles in cellular functions including signaling, transcription, translation, and stress response. Importantly, condensate misregulation is associated with human diseases, including neurodegeneration and cancer among others. When condensate-forming biomolecules are fluorescently-labeled and examined with fluorescence microscopy they appear as illuminated foci, or puncta, in cells. Puncta features such as number, volume, shape, location, and concentration of biomolecular species within them are influenced by the thermodynamics of biomolecular interactions that underlie LLPS. Quantification of puncta features enables evaluation of the thermodynamic driving force for LLPS and facilitates quantitative comparisons of puncta formed under different cellular conditions or by different biomolecules. Our work on nucleoporin 98 (NUP98) fusion oncoproteins (FOs) associated with pediatric leukemia inspired us to develop an objective and reliable computational approach for such analyses. The NUP98-HOXA9 FO forms hundreds of punctate transcriptional condensates in cells, leading to hematopoietic cell transformation and leukemogenesis. To quantify the features of these puncta and derive the associated thermodynamic parameters, we developed a live-cell fluorescence microscopy image processing pipeline based on existing methodologies and open-source tools. The pipeline quantifies the numbers and volumes of puncta and fluorescence intensities of the fluorescently-labeled biomolecule(s) within them and generates reports of their features for hundreds of cells. Using a standard curve of fluorescence intensity versus protein concentration, the pipeline determines the apparent molar concentration of fluorescently-labeled biomolecules within and outside of puncta and calculates the partition coefficient (K p ) and Gibbs free energy of transfer (ΔG Tr ), which quantify the favorability of a labeled biomolecule partitioning into puncta. In addition, we provide a library of R functions for statistical analysis of the extracted measurements for certain experimental designs. The source code, analysis notebooks, and test data for the Punctatools pipeline are available on GitHub: https://github.com/stjude/punctatools. Here, we provide a protocol for applying our Punctatools pipeline to extract puncta features from fluorescence microscopy images of cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Baggett, Medyukhina, Tripathi, Shirnekhi, Wu, Pounds, Khairy and Kriwacki.)

LAG3 is an inhibitory receptor that is highly expressed on exhausted T cells. Although LAG3-targeting immunotherapeutics are currently in clinical trials, how LAG3 inhibits T cell function remains unclear. Here, we show that LAG3 moved to the immunological synapse and associated with the T cell receptor (TCR)-CD3 complex in CD4 + and CD8 + T cells, in the absence of binding to major histocompatibility complex class II-its canonical ligand. Mechanistically, a phylogenetically conserved, acidic, tandem glutamic acid-proline repeat in the LAG3 cytoplasmic tail lowered the pH at the immune synapse and caused dissociation of the tyrosine kinase Lck from the CD4 or CD8 co-receptor, which resulted in a loss of co-receptor-TCR signaling and limited T cell activation. These observations indicated that LAG3 functioned as a signal disruptor in a major histocompatibility complex class II-independent manner, and provide insight into the mechanism of action of LAG3-targeting immunotherapies., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Germline TP53 splicing variants are uncommon, and their clinical relevance is unknown. However, splice-altering variants at exon 4-intron 4 junctions are relatively enriched in pediatric adrenocortical tumors (ACT). Nevertheless, family histories of cancer compatible with classic Li-Fraumeni syndrome are rarely seen in these patients. We used conventional and in silico assays to determine protein stability, splicing, and transcriptional activity of 10 TP53 variants at exon 4-intron 4 junctions and analyzed their clinical correlates. We reviewed public databases that report the impact of TP53 variants in human cancer and examined individual reports, focusing on family history of cancer. TP53 exon 4-intron 4 junction germline variants were identified in 9 of 75 pediatric ACTs enrolled in the International Pediatric Adrenocortical Tumor Registry and Children's Oncology Group ARAR0332 study. An additional eight independent TP53 variants involving exon 4 splicing were identified in the Pediatric Cancer Genome Project ( n = 5,213). These variants resulted in improper expression due to ineffective splicing, protein instability, altered subcellular localization, and loss of function. Clinical case review of carriers of TP53 exon 4-intron 4 junction variants revealed a high incidence of pediatric ACTs and atypical tumor types not consistent with classic Li-Fraumeni syndrome. Germline variants involving TP53 exon 4-intron 4 junctions are frequent in ACT and rare in other pediatric tumors. The collective impact of these germline TP53 variants on the fidelity of splicing, protein structure, and function must be considered in evaluating cancer susceptibility. IMPLICATIONS: Taken together, the data indicate that splice variants at TP53 codon 125 and surrounding bases differentially impacted p53 gene expression and function., (©2021 American Association for Cancer Research.)