Polymerases ε and δ maintain genome integrity through exonuclease proofreading. Germline and somatic pathogenic variants (PVs) in the exonuclease domain (ED) of POLE and POLD1 impair proofreading, causing hypermutated tumors. Despite shared mutational features that make these tumors highly immunogenic, molecular and clinical distinctions between POLE and POLD1 mutations and between somatic and germline variants remain incompletely understood. We compared the molecular and clinical characteristics of POLE and POLD1 ED PVs (n = 31), assessing their location, pathogenicity, clinical phenotypes, mismatch repair (MMR) status, tumor mutational burden, and signatures. We analyzed 360 proofreading-deficient tumors (source: The Cancer Genome Atlas [TCGA] and Catalogue Of Somatic Mutations In Cancer [COSMIC]) and 70 families (249 individuals) with polymerase proofreading-associated polyposis. All germline and somatic PVs had high AlphaMissense scores (0.87-1) and clustered within or near Exo motifs. Recurrent, nonfounder germline PVs, POLE L424V and POLD1 S478N, showed low/modest REVEL scores. Somatic variants occurred mainly in endometrial cancers (75% of proofreading-deficient TCGA cancers), whereas colorectal cancer predominated in polymerase proofreading-associated polyposis (56% of carriers). Cancer risks and tumor spectra differed between POLE and POLD1 PV carriers. Aggressive hereditary phenotypes were linked to either specific POLE PVs (eg, S297F, V411L, P436R, M444K, A456P, and S461T) or the co-occurrence of germline ED PVs with germline MMR gene PVs. Distinct hypermutator profiles were confirmed for polymerase ε and polymerase δ proofreading deficiencies via unique mutational signatures (Polymerase ε: SBS10a/b, SBS28; Polymerase δ: SBS10c/d). Tumors with combined proofreading and MMR deficiencies had significantly higher tumor mutational burden and a shift in the associated mutational spectra. Unlike POLE, POLD1 ED PVs exhibited haplosufficiency, typically requiring a somatic second hit (eg, loss of heterozygosity) or MMR deficiency to drive hypermutation. In conclusion, differences between POLE and POLD1 and between somatic and germline mutations influence clinical presentation, mutagenic potential, and reliance on cooperating defects in tumorigenesis. These insights advance the understanding of proofreading-deficient cancers, with implications for diagnostics, genetic counseling, and precision oncology.

Here, we predicted that Plasmepsin II (PlmII) can explore open-flap conformations not sampled for human aspartic proteases: Cathepsin D, Renin, and Pepsin were used in molecular dynamics simulations. We combined 24 independent (50 ns) MD runs to improve the conformational sampling of each system. We discovered two PlmII noncompetitive selective inhibitors: SPB07935 and RH01201, with Ki values in the μM range by targeting the open-flap conformations. Both compounds did not inhibit human Cathepsin D (hCatD) at high concentrations. We predicted that SPB07935 and RH01201 bind stably to the flap cryptic pocket, keeping this hairpin in an open or semiopen conformation along the MD simulations, respectively. Significantly, SPB07935 inhibited the P. falciparum chloroquine-resistant strain FcB1 growth in vitro, with an IC50 value of 8 μM while having a lower toxicity for HEK-293 human cells (CC50 = 189 μM).

KRAS mutations are responsible for a quarter of all lung adenocarcinomas. However, the molecular mechanisms linking these mutations and their frequent secondary dosage amplification to tumor formation are still not fully understood. While ample evidence supports a crucial role for the MAPK pathway in tumor development, the primary effectors targeted by this pathway remain largely unexplored. Here we identify the transcriptional repressor Capicua (CIC) as a key target inactivated by KRAS/MAPK signaling in lung adenocarcinoma. We show that genetic loss of CIC recapitulates the phenotypic consequences of amplified KRAS signaling. Genetic disruption of CIC suppressed the requirement for Kras allelic imbalances and accelerated the transformation of bronchiolar Club cells. We also demonstrate that restoring CIC repressor activity impaired proliferation of CIC-deficient tumor cells and reverted resistance to MAPK pathway inhibitors. These results highlight the key role of CIC during lung tumor formation and suggest that selective pressure for effective CIC inactivation favors secondary amplification of KRAS/MAPK signaling in tumor cells.

The development of hematopoietic cell lineages is a highly complex process governed by a delicate interplay of various transcription factors. The expression of these factors is influenced, in part, by epigenetic signatures that define each stage of cell differentiation. In particular, the formation of B lymphocytes depends on the sequential silencing of stemness genes and the balanced expression of interdependent transcription factors, along with DNA rearrangement. We have investigated the impact of Dido3 deficiency, a protein involved in chromatin status readout, on B cell differentiation within the hematopoietic compartment of mice. Our findings revealed significant impairments in the successive stages of B cell development. The absence of Dido3 resulted in remarkable alterations in the expression of essential transcription factors and differentiation markers, which are crucial for orchestrating the differentiation process. Additionally, the somatic recombination process, responsible for generation of antigen receptor diversity, was also adversely affected. These observations highlight the vital role of epigenetic regulation, particularly the involvement of Dido3, in ensuring proper B cell differentiation. This study reveals new mechanisms underlying disruptive alterations, deepening our understanding of hematopoiesis and may potentially lead to insights that aid in the development of therapeutic interventions for disorders involving aberrant B cell development.

The development of hematopoietic cell lineages is a highly complex process governed by a delicate interplay of various transcription factors. The expression of these factors is influenced, in part, by epigenetic signatures that define each stage of cell differentiation. In particular, the formation of B lymphocytes depends on the sequential silencing of stemness genes and the balanced expression of interdependent transcription factors, along with DNA rearrangement. We have investigated the impact of Dido3 deficiency, a protein involved in chromatin status readout, on B cell differentiation within the hematopoietic compartment of mice. Our findings revealed significant impairments in the successive stages of B cell development. The absence of Dido3 resulted in remarkable alterations in the expression of essential transcription factors and differentiation markers, which are crucial for orchestrating the differentiation process. Additionally, the somatic recombination process, responsible for generation of antigen receptor diversity, was also adversely affected. These observations highlight the vital role of epigenetic regulation, particularly the involvement of Dido3, in ensuring proper B cell differentiation. This study reveals new mechanisms underlying disruptive alterations, deepening our understanding of hematopoiesis and may potentially lead to insights that aid in the development of therapeutic interventions for disorders involving aberrant B cell development.

Germline activating variants in WWP1,which encodes an E3 ubiquitin ligase that antagonizes PTEN tumor suppressive function,have been proposed as an alternative mechanism of PTEN inactivation in PTEN-hamartoma-tumor syndrome(PHTS)-like patients with wildtype PTEN.1 More specifically,heterozygous,potentially activating WWP1 variants were first identified by Lee et al in patients affected with gastrointestinal oligopolyposis,including adenomatous,hyperplastic/serrated,and hamartomatous polyps,and occasionally with colorectal cancer(Table 1).Subse-quently,based on the PHTS phenotypic features,WWP1 mutational screening was performed in patients with thy-roid nodules,2 or normocephalic autism spectrum disorder(ASD),3 where germline WWP1 variants were also identified(Table S1).

The prevalence of overweight and obesity continues to rise in the population worldwide. Because it is an important predisposing factor for cancer, cardiovascular diseases, diabetes mellitus, and COVID-19, obesity reduces life expectancy. Adipose tissue (AT), the main fat storage organ with endocrine capacity, plays fundamental roles in systemic metabolism and obesity-related diseases. Dysfunctional AT can induce excess or reduced body fat (lipodystrophy). Dido1 is a marker gene for stemness; gene-targeting experiments compromised several functions ranging from cell division to embryonic stem cell differentiation, both in vivo and in vitro. We report that mutant mice lacking the DIDO N terminus show a lean phenotype. This consists of reduced AT and hypolipidemia, even when mice are fed a high-nutrient diet. DIDO mutation caused hypothermia due to lipoatrophy of white adipose tissue (WAT) and dermal fat thinning. Deep sequencing of the epididymal white fat (Epi WAT) transcriptome supported Dido1 control of the cellular lipid metabolic process. We found that, by controlling the expression of transcription factors such as C/EBPα or PPARγ, Dido1 is necessary for adipocyte differentiation, and that restoring their expression reestablished adipogenesis capacity in Dido1 mutants. Our model differs from other lipodystrophic mice and could constitute a new system for the development of therapeutic intervention in obesity.

Figure S2: Read alignments from WES-cfDNA, showing somatic mutations in APC and TP53 genes.

Table S5: List of KDR/VEGFR2 somatic mutations from the Cosmic, TCGA, GENIE and PCA-WGS databases.

Table S3: Treatment regimens of the Avatar patient derived xenograft models carrying the VEGFR2 WT and L840F genotypes.

Figure S5: A) Levels of phosphorylated ERK following treatment of the MDST8 colorectal cell line, expressing R1032Q VEGFR2, with different kinase inhibitors in the presence. B) Proliferation assays of Colo320 cell lines, stably expressing WT and R1032Q VEGFR2, upon treatment with TKIs. The expression of the VEGFR2 R1032Q hot-spot mutant in Colo320 cell lines (WT to KRAS/NRAS/BRAF/PIK3CA and mutated to TP53 and APC) increased sensitivity to cabozantinib.

Legends for Supplementary Figures 1-6

Table S2: Custom primers and probes designed based on the Thermofisher online design tool for TaqManÃ,® genotyping assay for KDR c.2518C>T (VEGFR2 p.L840L). TaqManÃ,® MGB (minor groove binder) probes incorporate a 5' reporter (VIC or FAM) and a 3' nonfluorescent quencher (NFQ).

Table S1: Mutation analysis concordance between plasma cfDNA exome sequencing and tumor exome sequencing.

The Kunitz‐Soybean Trypsin Inhibitor (Kunitz‐STI) family is a large family of proteins with most of its members being protease inhibitors. The versatility of the inhibitory profile and the structural plasticity of these proteins, make this family a promising scaffold for designing new multifunctional proteins. Historically, Kunitz‐STI inhibitors have been classified as canonical serine protease inhibitors, but new inhibitors with novel inhibition mechanisms have been described in recent years. Different inhibition mechanisms could be the result of different evolutionary pathways. In the present work, we performed a structural analysis of all the crystallographic structures available for Kunitz‐STI inhibitors to characterize serine protease‐binding loop structural features and locations. Our study suggests a relationship between the conformation of serine protease‐binding loops and the inhibition mechanism, their location in the β‐trefoil fold, and the plant source of the inhibitors. The classical canonical inhibitors of this family are restricted to plants from the Fabales order and bind their targets via the β4–β5 loop, whereas serine protease‐binding loops in inhibitors from other plants lie mainly in the β5–β6 and β9–β10 loops. In addition, we found that the β5–β6 loop is used to inhibit two different families of serine proteases through a steric blockade inhibition mechanism. This work will help to change the general perception that all Kunitz‐STI inhibitors are canonical inhibitors and proteins with protease‐binding loops adopting noncanonical conformations are exceptions. Additionally, our results will help in the identification of protease‐binding loops in uncharacterized or newly discovered inhibitors, and in the design of multifunctional proteins.

Background Glioblastoma is the most common and devastating primary brain cancer. Radiotherapy is standard of care; however, it is associated with brain radiation toxicity (BRT). This study used a multi-omics approach to determine whether BRT-related genes (RGs) harbor survival prognostic value and whether their encoded proteins represent novel therapeutic targets for glioblastoma. Methods RGs were identified through analysis of single-nucleotide variants associated with BRT (R-SNVs). Functional relationships between RGs were established using Protein-Protein Interaction networks. The influence of RGs and their functional groups on glioblastoma prognosis was evaluated using clinical samples from the Glioblastoma Bio-Discovery Portal database and validated using the Chinese Glioma Genome Atlas dataset. The identification of clusters of radiotoxic and putative pathogenic variants in proteins encoded by RGs was achieved by computational 3D structural analysis. Results We identified the BRT-related 15CAcBRT molecular signature with prognostic value in glioblastoma, by analysis of the COMT and APOE protein functional groups. Its external validation confirmed clinical relevance independent of age, MGMT promoter methylation status, and IDH mutation status. Interestingly, the genes IL6, APOE, and MAOB documented significant gene expression levels alteration, useful for drug repositioning. Biological networks associated with 15CAcBRT signature involved pathways relevant to cancer and neurodegenerative diseases. Analysis of 3D clusters of radiotoxic and putative pathogenic variants in proteins coded by RGs unveiled potential novel therapeutic targets in neuro-oncology. Conclusions 15CAcBRT is a BRT-related molecular signature with prognostic significance for glioblastoma patients and represents a hub for drug repositioning and development of novel therapies.

Figure S4: A) Molecular dynamics simulations with L840F VEGFR2 indicates that most of the adopted F840 conformations during the simulation are not compatible with inhibitor binding. Clashes between VEGFR2 and the sorafenib area are shown as red discs. B) Kinase assays showing impaired kinase activities of VEGFR2 L840F and R1032Q compared to wild-type. C) Effects of the L840F mutation on Y1175 VEGFR2 phosphorylation. HEK293 cells were transiently transfected with increasing levels of a plasmid encoding for WT or L840F VEGFR2. Cells at 70% confluence were starved in 1% BSA/DMEM for 4 h, and then incubated in the absence or presence of 60 ng/ml VEGF165 for 10 min at 37 {degree sign}C. Whole cell lysates were analyzed by western blotting, using antibodies against phosphoY1175 and total VEGFR2. Representative results are shown. D) PAE cells stably expressing WT or L840F VEGFR2 were generated from a PAE cell line that does not normally express VEGFR2 (empty). Cells at 70% confluence were starved in 1% BSA/DMEM for 4h, and then incubated in the absence or presence of 60 ng/ml VEGF165 for 10 min at 37 {degree sign}C. Whole cell lysates were then analyzed by western blotting, using antibodies against phosphoY1175 and total VEGFR2. Representative results are shown, highlighting the significant decrease in VEGF-induced Y1175 VEGFR2 phosphorylation in the presence of the L840F mutation.

Table S4: Primers used for site-directed mutagenesis. The mutated nucleotides are indicated in bold and underlined in each mutant.

Figure S6: Assessment of the expression of phosphorylated ERK (p-ERK) in the WT and L840F VEGFR2 PDX Avatar models by immunohistochemistry (A), confocal microscopy (B), and immunoblotting (C). The MAPK pathway was activated in the L840F VEGFR2 mutant model and was not decreased following treatment with TKIs.