Mutations of EIF2AK4 , which encodes the eIF2α kinase GCN2, cause a severe inherited form of pulmonary hypertension called pulmonary veno-occlusive disease (PVOD). Some pathogenic variants of GCN2 are amenable to pharmacological reactivation by low concentrations of ATP-pocket binding inhibitors. Kinase inhibition at modestly elevated concentrations limits the clinical utility of these drugs against PVOD. We therefore performed an in cellulo chemical screen for GCN2 activators and identified three structurally distinct compounds with low micromolar stimulatory activities. Unlike previously described GCN2 activators, one of these molecules activated GCN2 independently of GCN1. Modelling supported by structure activity screens suggested it binds within the ATP-pocket of GCN2, but unlike existing ligands does not protrude inward into the allosteric pocket or outward into the solvent. This overcomes a key requirement of other GCN2 activators.

The non-covalent interactions that underpin major cellular functions depend on molecular motion within 3D environments. Large depth-of-field single-molecule localization microscopy (3D-SMLM) methods facilitate these measurements, but their increased optical complexity and bespoke post-processing pipelines often sacrifice important cellular context. Here, we combine single-molecule light-field microscopy (SMLFM) with widefield Fourier light-field microscopy for correlative volumetric organelle imaging. The instantaneous acquisition of subcellular volumes improves the sensitivity of molecular organization, chemical environment, and diffusion measurements through the use of volumetric sub-cellular segmentation. We first demonstrate our approach by measuring the molecular organization of a nuclear-localized HaloTag protein relative to cell nuclei. Next, we characterize the molecular diffusion of the soluble protein, calreticulin, in the context of α 1 $\ualpha_1$ -antitrypsin deficiency, which revealed an increase in heterogeneous motion within endoplasmic reticulum inclusions.

Inhibitors of the protein kinase WEE1 have emerged as promising agents for cancer therapy. In this study, we uncover synergistic interactions between WEE1 small-molecule inhibitors and defects in mRNA translation, mediated by activation of the integrated stress response (ISR) through the kinase GCN2. Using a pooled CRISPRi screen, we identify GSPT1 and ALKBH8 as factors whose depletion confer hypersensitivity to the WEE1 inhibitor, AZD1775. We demonstrate that this synergy depends on ISR activation, which is induced by the off-target activity of WEE1 inhibitors. Furthermore, PROTAC-based WEE1 inhibitors and molecular glues show reduced or no ISR activation, suggesting potential strategies to minimise off-target toxicity. Our findings reveal that certain WEE1 inhibitors elicit dual toxicity via ISR activation and genotoxic stress, with ISR activation being independent of WEE1 itself or cell-cycle status. This dual mechanism highlights opportunities for combination therapies, such as pairing WEE1 inhibitors with agents targeting the mRNA translation machinery. This study also underscores the need for more precise WEE1 targeting strategies to mitigate off-target effects, with implications for optimising the therapeutic potential of WEE1 inhibitors. ### Competing Interest Statement The authors have declared no competing interest.

Alveolar type 2 (AT2) cells maintain lung health by acting as stem cells and producing pulmonary surfactant. AT2 dysfunction underlies many lung diseases, including interstitial lung disease (ILD), in which some inherited forms result from the mislocalization of surfactant protein C (SFTPC) variants. Lung disease modeling and dissection of the underlying mechanisms remain challenging due to complexities in deriving and maintaining human AT2 cells ex vivo. Here, we describe the development of mature, expandable AT2 organoids derived from human fetal lungs which are phenotypically stable, can differentiate into AT1-like cells, and are genetically manipulable. We use these organoids to test key effectors of SFTPC maturation identified in a forward genetic screen including the E3 ligase ITCH, demonstrating that their depletion phenocopies the pathological SFTPC redistribution seen for the SFTPC-I73T variant. In summary, we demonstrate the development of a novel alveolar organoid model and use it to identify effectors of SFTPC maturation necessary for AT2 health.

Somatic variants accumulate in non-malignant tissues with age. Functional variants, leading to clonal advantage of hepatocytes, accumulate in the liver of patients with acquired chronic liver disease (CLD). Whether somatic variants are common to CLD from differing etiologies is unknown. We analyzed liver somatic variants in patients with genetic CLD from alpha-1 antitrypsin (A1AT) deficiency or hemochromatosis. We show that somatic variants in SERPINA1, the gene encoding A1AT, are strongly selected for in A1AT deficiency, with evidence of convergent evolution. Acquired SERPINA1 variants are clustered at the carboxyl terminus of A1AT, leading to truncation. In vitro and in vivo, C-terminal truncation variants reduce disease-associated Z-A1AT polymer accumulation and disruption of the endoplasmic reticulum, supporting the C-terminal domain swap mechanism. Therefore, somatic escape variants from a deleterious germline variant are selected for in A1AT deficiency, suggesting that functional somatic variants are disease-specific in CLD and point to disease-associated mechanisms.

Proteomics MeT5A isogenics and MPM cell lines

To assess the presence and CT features of pulmonary cysts (PCs) in patients with renal neoplasms (RN) as a hallmark of Birt-Hogg-Dubé syndrome (BHDS). Single institution retrospective study of all patients with histological RN between May 2014 and May 2020. Individuals with non-renal neoplasm, nephroblastoma, benign cysts, < 18 years old, or without thoracic CT were excluded. Demographics, history of smoking, pneumothorax and cutaneous fibrofolliculomas/trichodischomas, family history of pneumothorax or RN, and genetic testing were recorded. Number, location, distribution and morphology of PCs were assessed on thoracic CT. Differences between patients with positive (BHD+) and negative (BHD−) genetics were analysed. An independent cohort of 10 BHDS patients was added to calculate the diagnostic accuracy of cyst features. Of 1475 patients with RN, 127 (8.6

Biallelic mutations in eukaryotic translation initiation factor 2 α kinase 4, EIF2AK4 (which encodes general control nonderepressible 2, GCN2) underpin heritable forms of pulmonary veno-occlusive disease (PVOD), a rare and fatal form of pulmonary hypertension. The mechanisms linking these are mostly uncharacterised. We demonstrate for the first time that homozygous loss of gcn2 is sufficient to cause mild pulmonary hypertension in mice. Single-cell transcriptomics of mouse lungs identified adventitial fibroblasts as having the greatest GCN2-dependent transcriptional differences, implicating them as key players in this model of PVOD. The most significantly upregulated pathways in gcn2-/- adventitial fibroblasts were inflammatory. Therefore, we went on to demonstrate a pro-inflammatory phenotype in gcn2-/- mouse embryonic fibroblasts and gcn2-/- mice. In a novel murine model of pulmonary hypertension induced by exposure to mitomycin C, deletion of interleukin-6 rescued the pulmonary vascular phenotype. When chronically exposed to lipopolysaccharide, the pulmonary hypertensive phenotype of gcn2-/- mice is exaggerated. Genetic ablation of interleukin-6 completely rescues both the baseline and LPS-exaggerated pulmonary hypertensive phenotype. Targeting Il6 -dependent pathways may be useful in treating this deadly disease. ### Competing Interest Statement The authors have declared no competing interest.

Inhibitors of the protein kinase WEE1 have emerged as promising agents for cancer therapy. In this study, we uncover synergistic interactions between WEE1 small-molecule inhibitors and defects in mRNA translation, mediated by activation of the integrated stress response (ISR) through the kinase GCN2. Using a pooled CRISPRi screen, we identify GSPT1 and ALKBH8 as factors whose depletion confer hypersensitivity to the WEE1 inhibitor, AZD1775. We demonstrate that this synergy depends on ISR activation, which is induced by the off-target activity of WEE1 inhibitors. Furthermore, PROTAC-based WEE1 inhibitors and molecular glues show reduced or no ISR activation, suggesting potential strategies to minimise off-target toxicity. Our findings reveal that certain WEE1 inhibitors elicit dual toxicity via ISR activation and genotoxic stress, with ISR activation being independent of WEE1 itself or cell-cycle status. This dual mechanism highlights opportunities for combination therapies, such as pairing WEE1 inhibitors with agents targeting the mRNA translation machinery. This study also underscores the need for more precise WEE1 targeting strategies to mitigate off-target effects, with implications for optimising the therapeutic potential of WEE1 inhibitors.

Fig. S1. SNP6.0 analysis of parental MeT5A-BAP1+/+ cells.Fig. S2. Expression of catalytically active BAP1 in parental MeT5A.Fig. S3. Characterisation of BAP1 expression in gene-edited MeT5A.Fig. S4. Whole genome sequencing of gene-edited MeT5A.Fig. S5. Proliferative profiles for gene-edited MeT5A.Fig. S6. Enriched GO terms in gene-edited MeT5A SILAC-MS.Fig. S7. Enriched KEGG pathways in gene-edited MeT5A SILAC-MS include EMT.Fig. S8. Metabolite responses to BAP1 mutation in isogenic MeT5A.Fig. S9. Immunoblotting for differentially expressed metabolic enzymes identified by SILAC-MS in isogenic MeT5A cell lines.Fig. S10. Characterisation of BAP1-status for MPM cell panel.Fig. S11. Evaluating correlation between BAP1 and selected metabolic enzymes in a panel of MPM cell lines and the TCGA MESO pan-cancer dataset.Fig. S12. Validation of ASS1 response to BAP1 alteration.Fig. S13. Improved prognosis for epithelioid MPM patients with loss of nBAP1 and increased expression of ASS1.Fig. S14. Relationship between BAP1 and ASS1 transcripts in the TCGA Pan-Cancer datasets for other cancer types.Fig. S15. The influence of BAP1-status on response to inhibition of purine metabolism.Fig. S16. The influence of BAP1-status on response to ASS1 inhibition.

Phase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. Recent work has raised the intriguing prospect that phase transitions in proteins and lipids can be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granules to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induces a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system serves as a potential regulatory mechanism in RNA trafficking and offers an important template to understand other examples across the cell whereby biomolecular condensates closely juxtapose organellar membranes.

Supplementary Methods, References and List of Figures and Tables

Background:Idiopathic pulmonary fibrosis is a fatal lung disease of progressive lung parenchymal scarring caused by the aberrant response of an alveolar epithelium repeatedly exposed to injury. Understanding epithelial dysfunction has been hampered by the lack of physiological alveolar type 2 (AT2) cell models and defined disease triggers. Monogenic forms of familial pulmonary fibrosis (FPF) caused by toxic gain-of-function variants provide an opportunity to investigate early pathogenic events. One such variant, surfactant protein C (SFTPC)-I73T, abnormally localises within AT2 cells and causes their dysfunction. Methods:We used base editing of fetal lung-derived AT2 (fdAT2) organoids to create a heterozygous disease model of endogenous SFTPC-I73T expression. We also created an inducible overexpression system to interrogate temporal changes associated with SFTPC-I73T expression. We cultured fdAT2 both in 3D culture and at air-liquid interface to understand the importance of polarity cues and air exposure on disease phenotypes. Results:In our heterozygous endogenous expression system, we found that fdAT2 expressing SFTPC-I73T grew without a lumen and were unable to correctly polarise. SFTPC-I73T accumulated with time and caused gross enlargement of early endosomes, preventing correct apico-basal trafficking of multiple endosomally trafficked cargoes including polarity markers and cell adhesion proteins. This phenotype was exacerbated by air exposure and led to loss of epithelial monolayer integrity and abnormal wound healing after injury. Conclusion:Using endogenous gene editing for the first time in differentiated alveolar organoids, we have demonstrated that the pathogenic effects of SFTPC-I73T are mediated through endosomal dysfunction and abnormal epithelial organisation. This has important implications for AT2 function in vivo.

Birt-Hogg-Dubé syndrome (BHDS) is the most common monogenic cause of pneumothorax. Most affected families have pathogenic variants in the FLCN gene. Using large genomic registries (UK Biobank (UKB), 100,000 Genomes Project and East London Genes & Health) including >550 000 individuals, we demonstrate that the frequency of clinically validated loss-of-function FLCN variants is 1 in 2710 to 4190. While the lifetime risk of pneumothorax in FLCN mutation carriers in the UKB and a BHDS clinical cohort was substantial (28.4% and 37.3%, respectively, to age 65 years), the lifetime risk of renal cancer was significantly lower in UKB than in BHDS patients (1% vs 32.1%). These findings highlight the importance of clinical context in managing individuals with FLCN mutations.

Heart failure and associated cachexia is an unresolved and important problem. We report a new model of severe heart failure that consistently results in cachexia. Mice lacking the integrated stress response (ISR) induced eIF2α phosphatase, PPP1R15A, exhibit a dilated cardiomyopathy and severe weight loss following irradiation, whilst wildtype mice are unaffected. This is associated with increased expression of Gdf15 in the heart and increased levels of GDF15 in the circulation. We provide evidence that blockade of GDF15 activity prevents cachexia and slows the progression of heart failure. Our data suggests that cardiac stress mediates a GDF15 dependent pathway that drives weight loss and worsens cardiac function. We show relevance of GDF15 to lean mass and protein intake with patients with heart failure. Blockade of GDF15 could constitute a novel therapeutic option to limit cardiac cachexia and improve clinical outcomes in patients with severe systolic heart failure.