bims-pideca 2026-05-31 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2026–05–31
twenty-one papers selected by
Ralitsa Radostinova Madsen, MRC-PPU

(Phospho)proteomic Profiling Reveals Mutation-Specific Adaptive Signaling to PI3Kα Inhibition in PIK3CA Mutant Breast Epithelial Cells.
AcTor, a novel mTOR stimulator, potentiates ixazomib for the treatment of acute myeloid leukemia.
The proximal lipid phase of PI3K signaling is confined to the plasma membrane.
Atypical PI3Ks coordinate chemotaxis, signaling dynamics, and multicellular development in Dictyostelium.
Morphological fingerprints enable machine learning based inference of neuroblastoma cell states without transcriptomics.
Targeted single-cell RNA and perturbation sequencing with TAP-seq.
SPIFEE -- A pipeline for analyzing traces of live-cell fluorescence microscopy data.
AKT1 glutarylation regulated by GCDH and SIRT5 suppresses oncogenic signaling.
Endothelial Klf9 fine-tunes Akt signaling to act as a transcriptional brake restraining retinal angiogenesis.
Tuning PI3K: striking the right balance for optimal CD8 T-cell responses.
Divergent signaling profiles in mTOR gain-of-function Smith-Kingsmore syndrome (SKS) and TSC2 deficiency.
OpenIMC: an open-source platform for analyzing single-cell and spatial proteomics by imaging mass cytometry.
Protocol for generating endogenous degron tags in essential transcription factors in human iPSCs via CRISPR-Cas9.
Sirolimus Treatment for Complex Fetal and Neonatal Vascular Anomalies: Emerging Evidence and Safety Considerations.
Subclonal IDH1/2 Mutations as a Targetable Vulnerability in Vascular Tumors.
NF1 and SPRED1/2 cooperate through RAS-MAPK-independent functions.
Practical statistics for bioimage analysis - a guide to experimental design and data interpretation.
Proteomics at scale: Bottlenecks and opportunities for early-career researchers in a fast developing field.
Safety analyses of the INAVO120 randomised phase III trial of inavolisib or placebo with palbociclib-fulvestrant in patients with PIK3CA-mutated, hormone receptor-positive, HER2-negative, endocrine-resistant advanced breast cancer.
A Far-Red FRET biosensor for AMPK enables multiplexed imaging of single-cell bioenergetic homeostasis.
MiTo: tracing the phenotypic evolution of somatic cell lineages via mitochondrial single-cell multi-omics.

J Proteome Res. 2026 May 28.

(Phospho)proteomic Profiling Reveals Mutation-Specific Adaptive Signaling to PI3Kα Inhibition in PIK3CA Mutant Breast Epithelial Cells.

Fujia Wang, Maarten Altelaar, Kelly E Stecker.

  Activating PIK3CA mutations are among the most frequent oncogenic drivers in breast cancer, with E545K and H1047R mutants representing the most prevalent hotspot variants. Despite the development of potent PI3K inhibitors, clinical efficacy remains limited in some cases. This underscores the need to understand how specific oncogenic PIK3CA mutations reshape signaling networks and therapeutic responses. Here, we compared the E545K and H1047R mutant breast epithelial cells to delineate mutation-specific signaling programs, growth phenotypes, and responses to PI3Kα inhibition in the presence and absence of insulin, using integrated growth assays and quantitative proteomic and phosphoproteomic profiling. These analyses uncovered mutation-specific signaling architectures and inhibitor sensitivities. Both mutants exhibited basal MAPK activation, but showed divergent MAPK phosphorylation dynamics in distinct PIK3CA mutations, suggesting a pivotal role for MAPK signaling. Upon PI3Kα inhibition with alpelisib, insulin engaged bypass signaling that partially counteracted downstream suppression. MEK inhibition alone suppressed the growth of PIK3CA mutant cells, and dual targeting of PI3K and MAPK signaling produced greater growth suppression than either single agent alone under insulin-stimulated conditions. Collectively, these findings reveal mutation-specific adaptive signaling and support combined PI3Kα and MAPK pathway inhibition as a strategy to improve therapeutic efficacy in PIK3CA mutant breast cancer.

Keywords:  MAPK Signaling; PIK3CA mutation; Phosphoproteomics, PI3K pathway; Proteomics

DOI:  https://doi.org/10.1021/acs.jproteome.5c00907
Mol Cancer. 2026 May 27.

AcTor, a novel mTOR stimulator, potentiates ixazomib for the treatment of acute myeloid leukemia.

Shakti P Pattanayak, Odai Darawshi, Omid Hajihassani, Jordan M Winter, Nicole Weiler, Melanie Ott, Florian Rothweiler, Jindrich Cinatl, Martin Michaelis, Daniel J Lindner, Thomas D Green, Polina Krassovskaia, Raphael T Aruleba, Kelsey H Fisher-Wellman, Jason A Mears, David Wald, Leif A Eriksson, Boaz Tirosh.

BACKGROUND: mTORC1 activity is oncogenic. However, in the presence of chemotherapy, suppression of mTORC1 is cytoprotective. mTOR suppression requires an intact tuberous sclerosis complex (TSC), composed of TSC1, TSC2 and TBC1D7. Small molecules that activate mTOR by blocking the TSC are lacking.
METHODS: We applied in silico docking and medicinal chemistry to generate AcTor, a potential first-of-its-kind TSC2 inhibitor. Because inhibition of TSC2 results in increased sensitivity to proteasome inhibitors, we combined AcTor and the proteasome inhibitor ixazomib (IXZ) in various cancer cell types.
RESULTS: Potentiation of cytotoxic activity of IXZ by AcTor was observed across multiple acute myeloid leukemia (AML) cell lines and primary patient samples. The combination triggered a collapse of mitochondrial respiratory capacity, loss of mitochondrial membrane potential, accumulation of ROS and apoptosis. These attributes increased in drug-resistant AML. Transcriptomic profiling revealed that AcTor alone induced anabolic and oxidative phosphorylation programs, whereas AcTor/IXZ redirected the signaling towards stress-associated and pro-apoptotic transcriptional states, including a p53 pathway signature. In vivo studies revealed reduction in AML burden, depletion of blasts and of leukemic stem cells, and retention of activity upon relapse. AcTor/IXZ was equally potent in a TP53-mutated patient-derived xenograft model, exceeding the efficacy of standard-of-care.
CONCLUSIONS: As a TSC2 inhibitor, AcTor should not be used alone in cancer. When combined with proteasome inhibitors, the pharmacodynamics of AcTor shifts towards the development of a mitochondrial catastrophe in AML, which is durable, broad range, agnostic to TP53 mutations and to the acquisition of resistance to common clinical anti-AML drugs.

DOI: https://doi.org/10.1186/s12943-026-02689-4
bioRxiv. 2026 May 12. pii: 2026.05.08.723799. [Epub ahead of print]

The proximal lipid phase of PI3K signaling is confined to the plasma membrane.

Morgan M C Ricci, Hemani Patel, Maria J Montoya, Kanishka L Jayasuriya, Andrew Rectenwald, Magdalene A K Motter, Rachel C Wills, Gerald R V Hammond.

Class I phosphoinositide 3-kinases (PI3Ks) generate the lipid second messengers PIP 3 and PI(3,4)P 2 to control diverse cellular processes including growth, metabolism, and survival. Although these signals are classically thought to arise at the plasma membrane, several recent studies have proposed that PI3K signaling is propagated from intracellular membranes along the endocytic pathway. Here, we combined genomic tagging of endogenous PI3K pathway enzymes with single-molecule imaging and sensitive lipid biosensors to define the spatial organization of PI3K signaling in living cells. We find that PI3K catalytic subunits are recruited to the plasma membrane but do not undergo detectable endosomal translocation during receptor activation. Consistently, PIP 3 and PI(3,4)P 2 accumulation is restricted to the plasma membrane, despite enrichment of lipid phosphatases along the endocytic pathway. Functional perturbation experiments further show that degradation of PI(3,4)P 2 occurs predominantly at the plasma membrane, indicating that both synthesis and termination of proximal lipid signals are spatially confined to this compartment. Together, these results resolve the subcellular localization of proximal PI3K signaling and support a model in which lipid second messenger production is restricted to the plasma membrane, with diversification of downstream pathway outputs occurring through redistribution of activated effector proteins rather than intracellular propagation of lipid signals.

DOI: https://doi.org/10.64898/2026.05.08.723799
bioRxiv. 2026 May 13. pii: 2026.05.11.724005. [Epub ahead of print]

Atypical PI3Ks coordinate chemotaxis, signaling dynamics, and multicellular development in Dictyostelium.

Laith Bahlouli, Erik Zhang, Eric Jung, Taylor Brentjens, Elizabeth Rose, Harrison Drebin, Ben Edwards, Isabella Wischik, Alyssa Huynh, Jacqueline Shen, Will Callahan, Kevin Zhangxu, Marc Edwards.

Phosphoinositide 3-kinase (PI3K) signaling regulates protrusion, polarity, membrane uptake, and multicellular development in Dictyostelium discoideum , but these functions have been interpreted largely through canonical Class I PI3Ks and PI(3,4,5)P₃ production. This framework does not fully explain how PI3K-dependent pathways attenuate Ras activity, organize PI(3,4)P₂-associated polarity states, support cAMP relay, or coordinate development. Here, we identify three atypical PI3K-family enzymes-PikF, PikG, and PikH-as functionally distinct regulators of these processes. PikF constrains Ras-phosphoinositide-actin signaling; pikF⁻ cells show prolonged cAMP-stimulated Ras activation, extended PIP₃ recruitment, delayed PI(3,4)P₂ biosensor recovery, elevated peripheral actin activity, impaired chemotactic precision, and delayed abnormal development. PikG acts through a distinct relay-associated pathway: pikG⁻ cells fail to generate endogenous cAMP oscillations, display disrupted ACA polarity, deposit spatially disorganized ACA-positive vesicle trails, and fail to aggregate. PikH, in contrast, supports efficient phagocytic uptake with little effect on acute chemotactic signaling. Kinase-dead rescue experiments show that conserved catalytic lysines are required for PikF- and PikG-dependent development and PikH-dependent uptake. Together, our results reveal that atypical PI3Ks diversify the Dictyostelium PI3K signaling toolkit, separating protrusive signal attenuation, cAMP relay organization, membrane uptake, and multicellular development into distinct kinase-dependent modules.

DOI: https://doi.org/10.64898/2026.05.11.724005
bioRxiv. 2026 May 13. pii: 2026.05.12.724731. [Epub ahead of print]

Morphological fingerprints enable machine learning based inference of neuroblastoma cell states without transcriptomics.

Vic Zamloot, Yuanzhong Pan, JinSeok Park.

Inference of cancer cell states is essential for understanding oncogenic mechanisms and predicting clinical outcomes, yet current reliance on transcriptomic profiling limits scalability and real-time monitoring. Here, we show that cell morphology provides a low-dimensional, observable representation of cellular identity and its dynamics. Using neuroblastoma (NB) as a model system, we establish a machine learning- morphology profiling framework that infers adrenergic (ADRN) and mesenchymal (MES) cell states directly from high-dimensional morphological fingerprints without reliance on transcriptomic measurements. By benchmarking against single-cell RNA sequencing (scRNA-seq), we demonstrate that morphology-defined states closely align with transcriptomic profiles at single-cell resolution. We further show that cell state transitions are represented as continuous trajectories within a morphology-defined state space. Perturbations targeting distinct regulatory layers, including ROCK signaling and epigenetic regulation via EZH2, drive convergent trajectories along a shared phenotypic axis. Together, these results establish cell morphology as a scalable and non-destructive readout of cell state with machine learning providing a unified framework for high-throughput phenotyping and real-time tracking of cancer cell state plasticity.

DOI: https://doi.org/10.64898/2026.05.12.724731
Nat Protoc. 2026 May 26.

Targeted single-cell RNA and perturbation sequencing with TAP-seq.

Dewi P I Moonen, Daniel Schraivogel, Andreas R Gschwind, Lars M Steinmetz.

Pooled genome editing combined with single-cell RNA sequencing-commonly known as Perturb-seq-has transformed the ability to interrogate genome function. However, whole-transcriptome single-cell RNA sequencing requires high sequencing depth to achieve the sensitivity needed for functional genomics screens, limiting its widespread use owing to prohibitive cost. Here we describe a detailed and updated protocol for targeted Perturb-seq (TAP-seq), a method that addresses the sensitivity and cost limitations of Perturb-seq. Instead of capturing the whole transcriptome, TAP-seq focuses on quantifying hundreds of transcripts of interest. The TAP-seq workflow involves first selecting genes for targeted readout, designing primers, conducting an initial pilot experiment and finally performing the TAP-seq screen and analyzing the data. We provide comprehensive guidance on designing targeted readout strategies for TAP-seq and describe all steps of the protocol, starting with library preparation. The outcome of TAP-seq is single-cell measurements of selected gene and guide RNA expression levels, guide RNA assignments to individual cells and differential expression results revealing perturbation effects on target genes. We further include instructions for adapting TAP-seq to all currently available single-cell RNA-sequencing platforms. Prior experience in single-cell technologies is beneficial and the protocol described can be completed in 2 days (excluding data analysis). In summary, this protocol describes how to perform sensitive, scalable and cost-effective single-cell perturbation screens.

DOI: https://doi.org/10.1038/s41596-026-01367-5
bioRxiv. 2026 May 11. pii: 2026.05.06.723263. [Epub ahead of print]

SPIFEE -- A pipeline for analyzing traces of live-cell fluorescence microscopy data.

Colin Hogendorn, Ingrid R Aragon, Samuel Dallon, Eric Batchelor.

To properly respond to their environment, cells adjust the activity of key regulatory proteins and rates of gene expression. Methods to detect and quantify these forms of regulatory dynamics in living cells are of central importance for understanding cellular signaling events in both physiological and pathological conditions. Current technologies in this field make use of fluorescent probes to track cell signaling dynamics. Although these technologies have been used for decades, challenges remain. In particular, the segmentation, tracking, and interpretation of single cell dynamic data are time-consuming, prone to subjective errors, and often lacking in standardization across experiments. Here, we present SPIFEE, a data pipeline that uses experiment-dependent parameters to smooth noise and quantify key features of fluorescence data from time-lapse imaging studies. Processing data in this manner enhances and accelerates quantification of live-cell gene and protein expression, simplifies data analysis, and facilitates hypothesis generation.
Author Summary: Cells adjust protein activity and gene expression levels over time to respond to changes in their environment, a process referred to as cell signaling dynamics. Quantifying cell signaling dynamics in living cells often uses fluorescent probes, such as green fluorescent protein (GFP) and its spectral variants, to track changes in gene expression or protein activity over time. Challenges inherent in analyzing fluorescence data from single cells stem from biological and experimental noise, time-consuming quantification, and subjective errors. To address these challenges, we developed a computational tool called Signal Processing and Integrated Feature Extraction (SPIFEE). The pipeline improves the quality of fluorescence data analysis by reducing noise and extracting signal features in a way that is both intuitive and objective. The pipeline provides more accurate, rapid, and unbiased quantification of time-lapse microscopy data.

DOI: https://doi.org/10.64898/2026.05.06.723263
Cell Rep. 2026 May 28. pii: S2211-1247(26)00472-9. [Epub ahead of print]45(6): 117394

AKT1 glutarylation regulated by GCDH and SIRT5 suppresses oncogenic signaling.

Longchang Bai, Ruocen Liao, Yu Zhang, Yongshi Jia, Lei Shi, Hongen Xu, Liang Liang, Yu Feng, Chenfang Dong.

  Aberrant AKT1 activation is a hallmark of cancer, yet the molecular mechanisms regulating its activity remain incompletely understood. Here, we report that AKT1 undergoes glutarylation at evolutionarily conserved lysines K179 and K289. Mechanistically, AKT1 glutarylation is regulated by the metabolic enzymes GCDH and DHTKD1, which modulate glutaryl-CoA levels, and reversed by the deglutarylase SIRT5. Glutarylation at K179 disrupts the K179-E198 salt bridge and AKT1-ATP interactions, whereas K289 glutarylation perturbs ATP coordination and reduces PDK1-mediated phosphorylation, collectively inactivating AKT1. Functionally, AKT1 glutarylation inhibits cell proliferation and tumor growth, whereas GCDH promotes these oncogenic functions by suppressing glutarylation. Notably, pharmacological MYC inhibition, which downregulates GCDH and elevates AKT1 glutarylation, synergizes with the AKT inhibitor afuresertib to suppress gastric cancer cell growth, revealing a potential therapeutic vulnerability. These findings link lysine metabolism to AKT-driven cancer progression and suggest therapeutic strategies targeting glutarylation dynamics.

Keywords:  AKT1; CP: Cancer; CP: Molecular biology; DHTKD1; GCDH; SIRT5; glutarylation

DOI:  https://doi.org/10.1016/j.celrep.2026.117394
Int J Biol Sci. 2026 ;22(10): 5203-5227

Endothelial Klf9 fine-tunes Akt signaling to act as a transcriptional brake restraining retinal angiogenesis.

Honglian Wu, Tianjing Yang, Yuning Xun, Ming Zhou, Yutian Zhang, Xueming Yao, Yongsheng Chang, Yi Lei, Hua Yan.

  Retinal angiogenesis requires precise transcriptional regulation. Krüppel-like factor 9 (Klf9) has been implicated in various biological processes; however, its specific role in retinal vascular development and ocular neovascular disease remains unclear. In this study, we identified Klf9 as a critical transcriptional regulator of retinal vascular homeostasis. Spatiotemporal transcriptomic and single-cell RNA sequencing analyses revealed that Klf9 was highly enriched in retinal endothelial cells and upregulated during vascular maturation. Using genetic mouse models, we demonstrated that endothelial-specific Klf9 deletion accelerated neonatal retinal vascular expansion and tip cell formation, whereas its overexpression delayed angiogenesis and disrupted barrier function. In oxygen-induced retinopathy, Klf9 loss exacerbated pathological neovascularization and leakage, while its overexpression conferred protection. Integrated RNA-seq and ATAC-seq profiling of human retinal microvascular endothelial cells revealed that Klf9 represses a network of genes involved in the PI3K-Akt pathway and focal adhesions. Key effectors, including AKT1, PTK2, and RAC1, were suppressed by reduced chromatin accessibility at their promoters. Both in vitro and in vivo rescue experiments confirmed that Akt activation reverses vascular hypoplasia caused by Klf9 overexpression, whereas Akt inhibition normalizes the hyper-angiogenic phenotype of the Klf9-deficient endothelium. Collectively, these findings establish Klf9 as a transcriptional brake on retinal angiogenesis, acting through chromatin-mediated suppression of the PI3K-Akt pathway, and provide new mechanistic insights and potential therapeutic targets for pathological retinal angiogenesis.

Keywords:  Akt signaling; Angiogenesis; Endothelial cells; Klf9

DOI:  https://doi.org/10.7150/ijbs.133293
J Immunol. 2026 May 14. pii: vkaf296. [Epub ahead of print]215(5):

Tuning PI3K: striking the right balance for optimal CD8 T-cell responses.

Jennifer L Cannons, Andrea C Pichler, Pamela L Schwartzberg.

  CD8 cytolytic T cells are key players in fighting viral infections and other intracellular pathogens. In response to signals from the TCR, costimulatory molecules, and cytokines, CD8 T cells differentiate into populations of cytolytic effectors that can efficiently kill infected and tumor cells, as well as memory precursors that can develop into memory cells that respond to subsequent infection. The class I phosphatidylinositol 3-kinases (PI3Ks) are key components of signaling pathways that dictate and shape CD8 T-cell responses during acute viral infection, as well as during CD8 T-cell exhaustion, a state of hyporesponsiveness driven by prolonged antigen exposure in chronic infection and cancer. In this review, we highlight the role of PI3Kδ in CD8 T-cell activation, differentiation, and function, with a focus on downstream effectors and lessons learned from activating and inhibitory mutants affecting these pathways in mouse models and primary immunodeficiencies.

Keywords:  PI3K; T-cell memory; cytotoxic CD8 T cells; exhaustion

DOI:  https://doi.org/10.1093/jimmun/vkaf296
bioRxiv. 2026 May 13. pii: 2026.05.09.724025. [Epub ahead of print]

Divergent signaling profiles in mTOR gain-of-function Smith-Kingsmore syndrome (SKS) and TSC2 deficiency.

Curtis R Carlson, Yang Shen, Hongzhi He, Erwin K Gudenschwager, Chunyan Hou, Junfeng Ma, Joanna C Chiu, Andrew C Liu.

Smith-Kingsmore syndrome (SKS) is a rare neurodevelopmental disorder caused by gain-of-function mutations in MTOR , yet whether these mutations phenocopy TSC2 loss or establish a distinct signaling state remains unclear. Using quantitative proteomics, phosphoproteomics, and transcriptomics in isogenic cell models of SKS ( MTOR Δ4aa ), TSC2 loss ( TSC2 -/- ), and wild-type controls under glucose depletion and refeeding, we find that MTOR Δ4aa and TSC2 -/- cells occupy fundamentally distinct regulatory states. TSC2 -/- cells exhibit broad anabolic remodeling and a transcriptional program dominated by NF-κB- and STAT-driven inflammatory responses. MTOR Δ4aa cells instead display enrichment of nuclear and RNA processing programs, E2F/MYC-driven transcription, and a constrained proteomic dynamic range across nutrient states. Phosphoproteomic analysis of MTOR Δ4aa reveals rerouting of nutrient-responsive signaling toward MAPK/ERK- and Ca 2+ /CaMK-dependent pathways with limited canonical mTORC1/S6K1 engagement. These findings establish SKS as a signaling rewiring disorder distinct from classical mTORC1 hyperactivation, with implications for therapeutic targeting.

DOI: https://doi.org/10.64898/2026.05.09.724025
Res Sq. 2026 May 12. pii: rs.3.rs-9558630. [Epub ahead of print]

OpenIMC: an open-source platform for analyzing single-cell and spatial proteomics by imaging mass cytometry.

Dean Tessone, Mohamed Kamal, Valerie Hennes, James Hicks, Peter Kuhn.

Imaging Mass Cytometry (IMC) enables spatially resolved single-cell proteomics, but fragmented data analysis tools limit reproducibility. We present OpenIMC, an open-source platform consolidating IMC workflows into a cohesive environment. OpenIMC integrates visualization, preprocessing, segmentation, feature extraction, phenotyping, and spatial analysis. Accessible via graphical and command-line interfaces sharing a backend, the platform ensures consistent execution. Built for scalability and reproducibility, OpenIMC automatically records analytical parameters and enables sharing of complete sessions. Case studies analyzing blood-derived cells and tissue slices validate the software platform by recovering known phenotypes and structural organization. OpenIMC lowers technical barriers, supporting rigorous, scalable single-cell and spatial proteomics.

DOI: https://doi.org/10.21203/rs.3.rs-9558630/v1
STAR Protoc. 2026 May 28. pii: S2666-1667(26)00255-8. [Epub ahead of print]7(2): 104602

Protocol for generating endogenous degron tags in essential transcription factors in human iPSCs via CRISPR-Cas9.

Ziwei Li, Arthur Roberts, Utkal Nimse, Lucas Vu, Jia Fei.

  Precise genome engineering in human pluripotent stem cells remains inefficient, limiting endogenous fluorescent tagging needed to study phase-separated membraneless nuclear compartments. Here, we present a protocol for generating precise knockin alleles of essential genes in human induced pluripotent stem cells (iPSCs) via CRISPR-Cas9 editing. We describe steps for combining transient p53 inhibition, optimized transfection conditions, and fluorescence-activated cell sorting. This protocol enables recovery of viable edited clones that would otherwise be lost due to editing-induced stress or essential gene perturbation. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Keywords:  Cell Biology; Genomics; Molecular Biology

DOI:  https://doi.org/10.1016/j.xpro.2026.104602
J Clin Med. 2026 May 13. pii: 3739. [Epub ahead of print]15(10):

Sirolimus Treatment for Complex Fetal and Neonatal Vascular Anomalies: Emerging Evidence and Safety Considerations.

Elena Țarcă, Elena Cojocaru, Lăcrămioara Ionela Butnariu, Solange Tamara Roșu, Alina Costina Luca, Sidonia Susanu, Vasile Valeriu Lupu, Eduard Vasile Roșu, Paula Popovici, Viorel Țarcă.

  Background: Vascular abnormalities (VAs) are a broad category of disorders that include vascular malformations (inadequate embryologic blood vessel development) and vascular tumors (characterized by vascular cell hyperplasia). This sometimes-complicated pathology is increasingly being treated with mTOR inhibitors, particularly sirolimus (rapamycin). Only a limited number of neonates treated with sirolimus have been reported in the literature to date, and there is no consensus regarding the optimal treatment regimen for vascular anomalies in this population. The objective of our narrative review is to summarize the most recent information from the specialized literature regarding the efficacy and safety of sirolimus in newborns. Methods: A methodological search was performed through the available data concerning the indications and safety when using sirolimus to treat VAs in newborns. We evaluated articles from the Embase and PubMed academic search engines using the following search terms: (vascular anomalies OR vascular tumors OR venous malformations OR lymphatic malformations OR capillary malformations) AND (neonate) AND (sirolimus OR rapamycin). Results: We identified a lack of consensus regarding indications for treatment initiation, optimal dosing regimens, and duration of therapy. In most published studies, neonates were analyzed within broader pediatric cohorts that included patients up to 18 years of age, rather than as a distinct population. Moreover, only a limited number of investigations have specifically focused on neonates treated with sirolimus and evaluated outcomes from a neonatal subgroup perspective. The available evidence largely consists of isolated case series and reports describing sirolimus use in fetuses and neonates, most commonly for lymphatic malformations or kaposiform hemangioendothelioma; in some instances, sirolimus was administered in combination with propranolol, corticosteroids, or other therapeutic modalities. Nevertheless, across studies and case reports involving fetuses or neonates, sirolimus is generally reported to be effective and well tolerated, with adverse effects that are minimal and reversible. Conclusions: For neonates and even fetuses with large, complex VAs, sirolimus appears to be an effective treatment with no serious adverse events reported to date. Decisions regarding off-label sirolimus initiation should be made by a multidisciplinary team, and parents must be thoroughly informed about potential adverse events. Well-designed randomized controlled trials or high-quality observational studies are needed to further evaluate the efficacy and safety of sirolimus in the neonatal population.

Keywords:  neonate; rapamycin; sirolimus treatment; vascular abnormalities

DOI:  https://doi.org/10.3390/jcm15103739
bioRxiv. 2026 May 17. pii: 2026.05.14.725132. [Epub ahead of print]

Subclonal IDH1/2 Mutations as a Targetable Vulnerability in Vascular Tumors.

Dong-Min Yu, Eunice Lee, Gabriel J Starrett, Zili Zhai, Eleanor Dowell, Kaitlyn Walsh, Andrew Day, Doreen Palsgrove, Justin Bishop, Dylan M Marchione, Maryam Asgari, Stephen Chung, Whitney High, Joyce Mc Teng, Josh Wisell, Breelyn A Wilky, Donald A Glass, Gregory A Hosler, Richard C Wang.

Despite extensive sequencing, the genetic etiology of sporadic angiosarcoma remains poorly defined (1-3). Maffucci syndrome, characterized by vascular tumors and elevated cancer risk, is driven by mosaic gain-of-function mutations in IDH1/2 (4,5), though these have not been reported in sporadic angiosarcoma. We identify recurrent, low-variant allele frequency hotspot mutations in IDH1/2 in over half of sporadic angiosarcomas. Mutations were validated by Sanger sequencing and immunohistochemistry. Mutant IDH1 endothelial cells promote tumorigenesis through non-cell-autonomous mechanisms, secreting 2-hydroxyglutarate (2-HG) to increase growth factor and endothelial-to-mesenchymal transition gene expression, activate pAkt/pERK signaling, induce DNA methylation changes, and promote anchorage-independent growth, which are reversed by the mutant IDH1 inhibitor ivosidenib. Patients with mosaic IDH1 mutations show reduced serum 2-HG and marked tumor regression following ivosidenib treatment. The clinical efficacy of ivosidenib in vascular tumors with subclonal IDH1 mutations suggests that low VAF IDH1/2 mutations may be a targetable vulnerability in sporadic angiosarcoma. (6,7).
Statement of Significance: We identify recurrent, low-VAF IDH1/2 mutations in angiosarcoma and provide evidence that these subclonal mutations promote tumorigenesis through non-cell-autonomous mechanisms. Vascular tumors driven by subclonal IDH1 mutations responded dramatically to ivosidenib, thus revealing a novel treatment for a subset of vascular tumors.

DOI: https://doi.org/10.64898/2026.05.14.725132
Proc Natl Acad Sci U S A. 2026 Jun 02. 123(22): e2535319123

NF1 and SPRED1/2 cooperate through RAS-MAPK-independent functions.

Jillian M Silva, Lizzeth Canche, Alice Cheng, Lucy C Young, Frank McCormick.

  Neurofibromin, the protein product of the neurofibromatosis type 1 (NF1) gene, requires the direct binding interaction with SPRED to negatively regulate the RAS-MAPK pathway. Although the region of neurofibromin that stimulates the intrinsic GTPase activity of RAS represents only a small percentage of the entire protein, a large degree of the NF1 structural domains and their correlating mechanistic functions remain elusive. Here, we demonstrate RAS-independent biochemical and signaling functions regulated by the coordinate control of NF1 and SPRED1/2. Utilizing CRISPR-Cas9 methods to ablate NF1 or SPRED1/2 in isogenic "RASless" mouse embryonic fibroblast (MEF) cell lines expressing either the KRAS4b wild-type variant or an oncogenic KRAS-mutation, we show loss of SPRED1/2 phenocopies NF1 loss and their cooperation is required to modulate MAPK-AKT signaling. Moreover, NF1 or SPRED1/2 loss also resulted in a potent suppression of the RAS family GTPases, RRAS and RRAS2, occurring independently of RAS or AKT pathway activation. A transcriptome microarray analysis of the NF1 or SPRED1/2 knockout MEF cells revealed a specific subset of RAS-independent, NF1-SPRED1/2-dependent gene signatures, in which these same genes were also directly regulated by the RAS-GTPase function of neurofibromin. The modulation of these NF1-SPRED1/2-dependent downstream signaling effectors were further corroborated in Schwann cell models derived from Neurofibromatosis type I patients that consisted of either plexiform neurofibroma cells or unaffected nerve cells abrogated of NF1 or neurofibromin RAS-GAP activity. Taken together, this study provides RAS-independent functions that are dependent on the cooperation of NF1 and SPRED1/2 in a manner that is uncoupled from canonical MAPK signaling.

Keywords:  MAPK; RAS; SPRED; neurofibromin

DOI:  https://doi.org/10.1073/pnas.2535319123
J Cell Sci. 2026 May 15. pii: jcs264367. [Epub ahead of print]139(10):

Practical statistics for bioimage analysis - a guide to experimental design and data interpretation.

Stefania Marcotti, Lina Gerontogianni, Gavin Kelly, David J Barry.

  Bioimage analysis is a powerful tool for investigating complex biological processes, but its robustness depends on technical precision and rigorous experimental design. In particular, the use of appropriate controls and experimental repetition is critical for drawing meaningful conclusions. However, there are times when both are inadequately applied or overlooked in favour of 'statistical significance', often derived from misused or misinterpreted statistical tests. In this Perspective, we reanalyse publicly available image datasets to highlight the crucial role of robust experimental design in interpreting results. Our findings underscore the importance of focusing on effect sizes and biological relevance over arbitrary statistical thresholds. We also discuss the diminishing returns of increased data collection once statistical stability has been achieved. By refining control usage and emphasising effect sizes, this Perspective offers guidance to enhance the reproducibility and robustness of research findings. We provide open access code to allow researchers to engage with the dataset, promoting better practices in experimental design and data interpretation.

Keywords:  Bioimage analysis; Reproducibility; Statistics

DOI:  https://doi.org/10.1242/jcs.264367
J Proteomics. 2026 May 24. pii: S1874-3919(26)00082-5. [Epub ahead of print] 105679

Proteomics at scale: Bottlenecks and opportunities for early-career researchers in a fast developing field.

Young Proteomics Investigators Club. Electronic address: ypic@eupa.org

  The field of proteomics has rapidly evolved over the last five years enabled by rapid advances in instrumentation and computation. At the same time, the proteomics community is also growing. This is reflected by the increasing participation in international conferences such as those organized by the European Proteomics Association and the Human Proteome Organization. These events provide early-career researchers with unique opportunities to exchange ideas, develop collaborations, and build networks that support professional development. One such network is the Young Proteomics Investigators Club, a European initiative supported by European Proteomics Association and led by early-career researchers. In this Community-Driven project, we investigate recent trends in proteomics by screening conference abstracts and evaluating the session attendance at Human Proteome Organization Congresses and European Proteomics Association conferences. Based on these analyses, we identified five areas that, from our perspective, are shaping the current trends in proteomics: clinical proteomics, proteomics of post-translational modifications, single-cell proteomics, systems biology and multi-omics, and computational proteomics. For each area, we highlight both unique challenges and identify a common theme: a shift from exploratory studies with manageable sample numbers toward large screenings and cohorts and the generation of big data, which often comes with the lack of computational support, organizational networks, and infrastructure. In this light, we describe the unique challenges and opportunities faced by early-career researchers. We point to actionable directions for enabling reproducible and transparent proteomics as well as community-driven projects and initiatives, which are often providing training and support. SIGNIFICANCE: In this perspective, the Young Proteomics Investigators Club (YPIC) discusses advances in analytical developments and computational approaches in proteomics research. Based on empirical analysis of recent European Proteomics Association conference and Human Proteome Organization congresses contributions, we identify clinical, single-cell, post-translational and systems-level proteomics as the research areas that have gained most momentum in the last three to five years. What makes this work distinctive is that it is written by and for early-career researchers, thereby uniquely identifying where momentum, challenges, and unmet needs converge for the newest generation of proteomics researchers. Rather than cataloguing advances, we examine the widening gap between what modern proteomics can generate and what individual researchers can realistically process, validate, and interpret. We describe specific structural barriers including access to high performance computing, limited formal training in scalable data analysis, the need for unified benchmarking standards and navigating clinical collaboration frameworks. We then highlight opportunities for the field, such as community-curated benchmarks, interdisciplinary mentorship models, and shared computational infrastructure. By making these challenges explicit from an early-career researchers standpoint, we aim to inform how training, funding, and community initiatives can be shaped to support the next generation of proteomics researchers.

Keywords:  Computational proteomics; Early career researchers; Proteomics; Systems biology; YPIC

DOI:  https://doi.org/10.1016/j.jprot.2026.105679
ESMO Open. 2026 May 27. pii: S2059-7029(26)01677-7. [Epub ahead of print]11(6): 107735

Safety analyses of the INAVO120 randomised phase III trial of inavolisib or placebo with palbociclib-fulvestrant in patients with PIK3CA-mutated, hormone receptor-positive, HER2-negative, endocrine-resistant advanced breast cancer.

S-A Im, K Kalinsky, K L Jhaveri, S Loibl, N Turner, C Saura, P Schmid, S Loi, E Hamilton, N Karadurmus, S Wang, A A Awan, E M Ciruelos, C-F Chung, E Thanopoulou, N Shankar, S Lim, Y Jin, C Song, D Juric.

   BACKGROUND: INAVO120 (NCT04191499) demonstrated significantly improved progression-free/overall survival with inavolisib plus palbociclib-fulvestrant versus placebo plus palbociclib-fulvestrant in patients with PIK3CA-mutated, hormone receptor-positive, HER2-negative, endocrine-resistant advanced breast cancer. We provide comprehensive analyses of key selected adverse events and their management.
MATERIALS AND METHODS: Inavolisib was given 9 mg orally once daily on days 1-28 of each 28-day cycle; palbociclib, at 125 mg orally once daily on days 1-21; fulvestrant, at 500 mg intramuscularly on days 1 and 15 of cycle 1, and every ∼28 days thereafter. Supportive therapies were used as clinically indicated. Key selected adverse events assessed included hyperglycaemia, stomatitis/mucosal inflammation, rash and diarrhoea, graded per National Cancer Institute Common Terminology Criteria for Adverse Events v5.0.
RESULTS: Data cut-off was 29 September 2023; median follow-up was 21.3 months (range 0-43.1; inavolisib arm) and 21.5 months (0.1-40.3; placebo arm). Inavolisib dose interruptions, reductions and discontinuations due to hyperglycaemia were observed in 27.2%, 2.5% and 0.6% of patients, respectively; the median time to first onset was 7.0 days (range 2.0-955.0). Metformin was the most commonly used antihyperglycaemic. No patients in the prediabetic population discontinued inavolisib due to hyperglycaemia. Inavolisib dose interruptions, reductions and discontinuations due to rash were observed in 1.2%, 0.6% and 0% of patients, respectively; the median time to first onset was 29.0 days (range 1.0-952.0). Topical hydrocortisone was most commonly used. Inavolisib dose interruptions, reductions and discontinuations due to stomatitis/mucosal inflammation were observed in 9.9%, 3.7% and 0.6% of patients, respectively; the median time to first onset was 13.0 days (range 1.0-610.0). Dexamethasone mouthwash was most commonly used. Inavolisib dose interruptions, reductions and discontinuations due to diarrhoea were observed in 6.8%, 1.2% and 0% of patients respectively; the median time to first onset was 13.0 days (range 1.0-610.0). Loperamide was most commonly used. Data were largely comparable across regions and ages. Inavolisib's safety profile was consistent with that of long-term treated populations.
CONCLUSIONS: This analysis demonstrates the generally consistent, manageable and tolerable safety profile of inavolisib plus palbociclib-fulvestrant. Key selected adverse events were generally reversible and were controlled by concomitant medications and dose modifications.

Keywords:  PIK3CA; diarrhoea; hyperglycaemia; inavolisib; rash; stomatitis/mucosal inflammation

DOI:  https://doi.org/10.1016/j.esmoop.2026.107735
bioRxiv. 2026 May 18. pii: 2026.05.13.723899. [Epub ahead of print]

A Far-Red FRET biosensor for AMPK enables multiplexed imaging of single-cell bioenergetic homeostasis.

Nicholaus L DeCuzzi, Marion Hardy, Markhus B Cabel, Jason Hu, Christina Abbate, Michael Pargett, John G Albeck.

Metabolic homeostasis has been studied primarily at the tissue and organism level, identifying molecular control mechanisms such as the energy charge-sensing kinase AMPK. Feedback loops involving AMPK and other regulators align cellular ATP generation and consumption, determining energetic balance. Recent work has demonstrated surprising oscillatory dynamics in AMPK activity, revealing unidentified kinetic modulation in single-cell homeostatic behaviour. However, probing the kinetic mechanisms of intracellular feedback requires simultaneous observation of multiple energetic parameters, and such experiments are precluded by the shared wavelength band occupied by most metabolic biosensors. We have overcome this obstacle by constructing a red-shifted FRET-based AMPK activity biosensor, RAMPKAR2, that is comparable to existing FRET-based AMPK activity biosensors. Multiplexed imaging of RAMPKAR2 with PercevalHR, which detects ATP/ADP ratio, confirmed that the kinetics of AMPK activity and ATP/ADP ratio are tightly coupled, with a lag of less than 6 minutes at the single-cell level. Pairing of RAMPKAR with HYlight, which detects the glycolytic intermediate fructose 1,6-bisphosphate (FBP), revealed that glycolytic activity co-oscillates with AMPK, shifted by ∼1.5 hours, and that these oscillations are suppressed by sustained AMPK activity. Together these data advance a model in which temporally offset increases in glycolytic ATP supply and AMPK deactivation contribute to single-cell oscillations.

DOI: https://doi.org/10.64898/2026.05.13.723899
Nat Commun. 2026 May 25.

MiTo: tracing the phenotypic evolution of somatic cell lineages via mitochondrial single-cell multi-omics.

Andrea Cossa, Alberto Dalmasso, Guido Campani, Elisa Bugani, Chiara Caprioli, Noemi Bulla, Andrea Tirelli, Yinxiu Zhan, Pier Giuseppe Pelicci.

Mitochondrial single-cell lineage tracing has recently emerged as a scalable and non-invasive tool to trace somatic cell lineages. However, the reliability and resolution of this technology remains highly debated. Here, we present MiTo, a novel end-to-end framework for robust mitochondrial single-cell lineage tracing data analysis. Benchmarked against real-world datasets, MiTo outperforms state-of-the-art methods and baselines in data pre-processing and clonal inference. Applied to a time-resolved dataset of breast cancer evolution (>2,500 cells), MiTo accurately infers ground-truth cell lineages (ARI = 0.94) and cell state transitions, detects clonal fitness markers, and quantifies heritability of gene regulatory networks. Comparing alternative lineage markers, MiTo quantifies the resolution limit of existing mitochondrial single-cell lineage tracing systems, which currently enable reliable inference of coarse-grained cellular ancestries, but not high-resolution phylogenetic inference. In conclusion, this work provides robust tools and practical guidelines to dissect somatic evolution with single-cell multi-omics.

DOI: https://doi.org/10.1038/s41467-026-71607-5