Proteomic analysis of tumor cell nuclear expulsion reveals significant cell adhesion and RNA binding programs in extracellular chromatin.

Understanding mechanisms of cancer cell death and the resulting effect on disease progression is crucial in cancer biology and the insight will likely offer better options for therapeutic treatment. Nuclear expulsion occurs in apoptotic cancer cells in a peptidylarginine deiminase 4 (Padi4) dependent manner. The resulting tumor cell nuclear expulsion product (TuNEP) promotes the outgrowth of neighboring cancer cells through chromatin-bound protein complexes.

Transient APC/C inactivation by mTOR boosts glycolysis during cell cycle entry.

Mammalian cells entering the cell cycle favour glycolysis to rapidly generate ATP and produce the biosynthetic intermediates that are required for rapid biomass accumulation. Simultaneously, the ubiquitin-ligase anaphase-promoting complex/cyclosome and its coactivator CDH1 (APC/C) remains active, allowing origin licensing and blocking premature DNA replication. Paradoxically, glycolysis is reduced by APC/C through the degradation of key glycolytic enzymes, raising the question of how cells coordinate these mutually exclusive events to ensure proper cell division.

Expression of IMPACT curtails metabolic plasticity and augments NK cell killing to abrogate metastatic growth.

Given the propensity of aggressive epithelial tumors to form hepatic metastases, we performed an in vivo cDNA screen using the mouse liver and KRASG12D/TP53R273H pancreatic cells to identify the RNA binding protein GCN1 as integral component of hepatic outgrowth. RNAi experiments reveal that GCN1 triggers the ISR to activate serine, folate, and methionine biosynthetic pathways together with amino acid transporters, which act in concert to facilitate acquisition of metabolites and to restore redox homeostasis. Alongside activation of the ISR, we found that GCN1 also functions in the nucleus where it interacts with HNRNPK to suppress the expression of MHC-I molecules, and natural killer (NK) ligands.

Potent β-lactam-based tyrosyl-DNA phosphodiesterase 1 inhibitors identified by a virtual screen.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is an important therapeutic target. We recently reported several crystal structures of TDP1 with small molecules bound within the catalytic pocket. These molecules bind by forming hydrogen bonds with residues of the catalytic HKN motifs.

CRISPR-Based Gene Dependency Screens reveal Mechanism of BRAF Inhibitor Resistance in Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) is the most aggressive form of thyroid cancer. Despite recent advances in treating BRAFV600E-driven ATC, therapy resistance remains a significant challenge, often resulting in disease progression and death. Leveraging a focused CRISPR/KO screen in parallel with a CRISPR/activation screen, both tailored on response to BRAFV600E inhibitor treatment, we identified TAZ (encoded by the WWTR1 gene) deficiency as synthetically lethal with BRAF inhibitor in ATC.

NOTCH1 reverses immune suppression in small cell lung cancer through reactivation of STING.

Downregulation of antigen presentation and lack of immune infiltration are defining features of small cell lung cancer (SCLC) limiting response to immune checkpoint blockade (ICB). While a high MHC Class I, immune-inflamed subset benefits from ICB, underlying mechanisms of immune response in SCLC have yet to be elucidated. Here we show that in the landmark IMpower133 clinical trial high, but not low, NOTCH1 expression is significantly associated with longer survival with the addition of ICB to chemotherapy among ~80% of SCLC patients with neuroendocrine-enriched tumors (ASCL1-enriched, HR 0.

RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria.

Despite progress in understanding pre-mRNA splicing, the regulatory mechanisms controlling most alternative splicing events remain unclear. We developed CRASP-Seq, a method that integrates pooled CRISPR-based genetic perturbations with deep sequencing of splicing reporters, to quantitively assess the impact of all human genes on alternative splicing from a single RNA sample. CRASP-Seq identifies both known and novel regulators, enriched for proteins involved in RNA splicing and metabolism.

Cell cycle dependent methylation of Dam1 contributes to kinetochore integrity and faithful chromosome segregation.

The kinetochore, a megadalton structure composed of centromeric (CEN) DNA and protein complexes, is required for faithful chromosome segregation in eukaryotes. The evolutionarily conserved Dam1/DASH complex (Ska1 in metazoans) is one of the essential protein sub-complexes of the budding yeast kinetochore. Previous studies showed that methylation of lysine residue 233 in Dam1 by Set1 is important for haploid growth as mutation of lysine 233 to alanine results in lethality.

Allelic effects on KLHL17 expression underlie a pancreatic cancer genome-wide association signal at chr1p36.33.

Pancreatic Ductal Adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the U.S. Both rare and common germline variants contribute to PDAC risk.

Transfer RNA acetylation regulates in vivo mammalian stress signaling.

Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here, we investigate the impact of -acetylcytidine (acC), a highly conserved tRNA modification catalyzed by the essential acetyltransferase Nat10. By targeting Thumpd1, a nonessential adapter protein required for Nat10-catalyzed tRNA acetylation, we determine that loss of tRNA acetylation leads to reduced levels of tRNA, increased ribosome stalling, and activation of eIF2α phosphorylation.

Immunophenotype of CAR T cells and apheresis products predicts response in CD22 CAR T cell trial for B cell acute lymphoblastic leukemia.

Although CAR T cell therapy is increasingly used to treat relapsed B cell acute lymphoblastic leukemia (ALL), 20%-30% of patients do not respond, and few clinical predictors of response have been established, especially in the pediatric population. A deeper analysis of CAR T cell infusion products, along with the apheresis product used as the starting material for CAR T cell manufacturing, provides valuable insights for predicting clinical outcomes. We analyzed infusion products and CD4/8-selected T cell starting materials from pediatric and young adult patients on a single-center study with relapsed/refractory B cell ALL who were undergoing treatment with CD22 CAR T cells and evaluated differences between T cells from responders and non-responders (NCT023215612).

Identification of molecular determinants of gene-specific bursting patterns by high-throughput imaging screens.

Stochastic transcriptional bursting is a universal property of active genes. While different genes exhibit distinct bursting patterns, the molecular mechanisms that govern gene-specific stochastic bursting are largely unknown. We have developed a high-throughput-imaging-based screening strategy to identify cellular factors that determine the bursting patterns of native genes in human cells.

A 3D Self-Assembly Platform Integrating Decellularized Matrix Recapitulates In Vivo Tumor Phenotypes and Heterogeneity.

Three-dimensional (3D) in vitro cell culture models are invaluable tools for investigating the tumor microenvironment. However, analyzing the impact of critical stromal elements, such as extracellular matrix (ECM), remains a challenge. In this study, we developed a hydrogel-free self-assembly platform to establish ECM-rich 3D "MatriSpheres" to deconvolute cancer cell-ECM interactions.

Metabolic dependency mapping identifies Peroxiredoxin 1 as a driver of resistance to ATM inhibition.

Metabolic pathways fuel tumor progression and resistance to stress conditions including chemotherapeutic drugs, such as DNA damage response (DDR) inhibitors. Yet, significant gaps persist in how metabolic pathways confer resistance to DDR inhibition in cancer cells. Here, we employed a metabolism-focused CRISPR knockout screen and identified genetic vulnerabilities to DDR inhibitors.

Chemical tools to define and manipulate interferon-inducible Ubl protease USP18.

Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine protease primarily responsible for deconjugating the interferon-inducible ubiquitin-like modifier ISG15 from protein substrates. Here, we report the design and synthesis of activity-based probes (ABPs) that incorporate unnatural amino acids into the C-terminal tail of ISG15, enabling the selective detection of USP18 activity over other ISG15 cross-reactive deubiquitinases (DUBs) such as USP5 and USP14. Combined with a ubiquitin-based DUB ABP, the USP18 ABP is employed in a chemoproteomics screening platform to identify and assess inhibitors of DUBs including USP18.

PhpC transcription factor infiltrates heterochromatin to generate cryptic intron-containing transcripts crucial for small RNA production.

The assembly of repressive heterochromatin in eukaryotic genomes is crucial for silencing lineage-inappropriate genes and repetitive DNA elements. Paradoxically, transcription of repetitive elements within constitutive heterochromatin domains is required for RNA-based mechanisms, such as the RNAi pathway, to target heterochromatin assembly proteins. However, the mechanism by which heterochromatic repeats are transcribed has been unclear.

Multiplatform metabolomic interlaboratory study of a whole human stool candidate reference material from omnivore and vegan donors.

Introduction: Human metabolomics has made significant strides in understanding metabolic changes and their implications for human health, with promising applications in diagnostics and treatment, particularly regarding the gut microbiome. However, progress is hampered by issues with data comparability and reproducibility across studies, limiting the translation of these discoveries into practical applications.

Objectives: This study aims to evaluate the fit-for-purpose of a suite of human stool samples as potential candidate reference materials (RMs) and assess the state of the field regarding harmonizing gut metabolomics measurements.

Allelic effects on expression likely mediated by JunB/D underlie a PDAC GWAS signal at chr1p36.33.

Pancreatic Ductal Adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the U.S. Both rare and common germline variants contribute to PDAC risk.

Targeted sulfur(VI) fluoride exchange-mediated covalent modification of a tyrosine residue in the catalytic pocket of tyrosyl-DNA phosphodiesterase 1.

Developing effective inhibitors of the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) has been challenging because of the enzyme shallow catalytic pocket and non-specific substrate binding interactions. Recently, we discovered a quinolone-binding hot spot in TDP1's active site proximal to the evolutionary conserved Y204 and F259 residues that position DNA. Sulfur (VI) fluoride exchange (SuFEx) is a biocompatible click chemistry reaction that enables acylation of protein residues, including tyrosine.

Tyrosine phosphorylation of both STAT5A and STAT5B is necessary for maximal IL-2 signaling and T cell proliferation.

Cytokine-mediated STAT5 protein activation is vital for lymphocyte development and function. In vitro tyrosine phosphorylation of a C-terminal tyrosine is critical for activation of STAT5A and STAT5B; however, the importance of STAT5 tyrosine phosphorylation in vivo has not been assessed. Here we generate Stat5a and Stat5b tyrosine-to-phenylalanine mutant knockin mice and find they have greatly reduced CD8 T-cell numbers and profoundly diminished IL-2-induced proliferation of these cells, and this correlates with reduced induction of Myc, pRB, a range of cyclins and CDKs, and a partial G1→S phase-transition block.

Transfer RNA acetylation regulates in vivo mammalian stress signaling.

Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here we investigate the impact of N4-acetylcytidine (acC), a highly conserved tRNA modification, using a Thumpd1 knockout mouse model. We find that loss of Thumpd1-dependent tRNA acetylation leads to reduced levels of tRNA, increased ribosome stalling, and activation of eIF2α phosphorylation.

Genome-scale exon perturbation screens uncover exons critical for cell fitness.

CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces.

IRF4 requires ARID1A to establish plasma cell identity in multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy that exploits transcriptional networks driven by IRF4. We employ a multi-omics approach to discover IRF4 vulnerabilities, integrating functional genomics screening, spatial proteomics, and global chromatin mapping. ARID1A, a member of the SWI/SNF chromatin remodeling complex, is required for IRF4 expression and functionally associates with IRF4 protein on chromatin.