bims-scepro 2025-10-19 papers

bims-scepro

Biomed News

on Stem cell proteostasis

Issue of 2025–10–19
nine papers selected by
William Grey, University of York

The E3 ubiquitin ligase Trip12 semi-selectively attenuates Wnt signaling.
GABA produced by multiple bone marrow cell types regulates hematopoietic stem and progenitor cells.
ID2 suppresses multiple myeloma cell proliferation by repressing the activity of the transcription factor TCF3.
Intracellular pH dynamics respond to extracellular matrix stiffening and mediate vasculogenic mimicry through β-catenin.
Cysteine-reactive covalent chloro-N-acetamide ligands induce ferroptosis mediated cell death.
Mass Spectrometry-Based Proteomic Analysis of Lysosome-Enriched Fractions.
Small-molecule OPA1 inhibitors reverse mitochondrial adaptations to overcome therapy resistance in acute myeloid leukemia.
The E3 ubiquitin ligase ZNRF3 restricts WNT receptor complex activity by stimulating the selective degradation of WNT-engaged FZD.
Viable and Functional: Long-Term -80 °C Cryopreservation Sustains CD34+ Integrity and Transplant Success.

iScience. 2025 Oct 17. 28(10): 113575

The E3 ubiquitin ligase Trip12 semi-selectively attenuates Wnt signaling.

Jessica Ensing, Amber D Ide, Carla Gilliland, Visakuo Tsurho, Emma Rudisel, Isabella Caza, Amber N Stratman, Nathan J Lanning, Stephanie Grainger.

  Wnt signaling is critical for the development and maintenance of many cell lineages, including hematopoietic stem cells (HSCs). The tight regulation of Wnt signals is essential, as overactivation can drive tumorigenesis. Numerous Wnt ligands and Frizzled (Fzd) receptors exist, and the negative regulation of particular Fzd signals is the focus of this study. We previously identified the requirement of Wnt9a-Fzd9b pairing for early HSC proliferation. However, the mechanisms controlling activation and signal termination are unclear. Here, we show that the E3 ubiquitin ligase Trip12 (thyroid hormone receptor interactor 12) targets the third intracellular loop of Fzd9b at K437, promoting lysosomal degradation by destabilizing Fzd9b membrane expression. Our data further indicates that Trip12 is semi-selective for Fzd9b. The Trip12-mediated reduction of Fzd9b surface expression dampens Wnt9a/Fzd9b signaling, affecting HSC proliferation in zebrafish. Our findings reveal a receptor-specific regulatory mechanism, with implications for targeted Wnt pathway therapies.

Keywords:  Biochemistry; Cell biology

DOI:  https://doi.org/10.1016/j.isci.2025.113575
Stem Cell Reports. 2025 Oct 16. pii: S2213-6711(25)00281-4. [Epub ahead of print] 102677

GABA produced by multiple bone marrow cell types regulates hematopoietic stem and progenitor cells.

Cesi Deng, Adedamola Elujoba-Bridenstine, Ai Tang Song, Rylie M Ceplina, Casey J Ostheimer, Molly C Pellitteri Hahn, Cameron O Scarlett, Sahitya Saka, Xuan Pan, Owen J Tamplin.

  Hematopoietic stem and progenitor cells (HSPCs) maintain homeostasis of the blood system by balancing proliferation and differentiation. Many extrinsic signals in the bone marrow (BM) microenvironment that regulate this balance are still unknown. We report gamma-aminobutyric acid (GABA) metabolite produced in the BM as a regulator of HSPCs. Deletion of the genes encoding the glutamate decarboxylase enzymes (Gad1 and Gad2) that produce GABA in either B lineages or endothelial cells (ECs) led to a slight reduction in BM HSPCs but not GABA levels. However, simultaneous blockade of GABA production from both hematopoietic cells and ECs resulted in a greater reduction of HSPCs and a significant reduction of BM GABA levels. Lower GABA levels in the BM altered the gene expression profile of HSPCs, with expression reduced for proliferation-associated genes and increased for B lineage genes. Our findings suggest GABA from multiple sources coordinates to regulate HSPC activity.

Keywords:  B cell lineage commitment; B cells; GABA; bone marrow niche; endothelial cells; gamma-aminobutyric acid; glutamate decarboxylase; hematopoietic stem and progenitor cells; microenvironment

DOI:  https://doi.org/10.1016/j.stemcr.2025.102677
Blood Cancer Discov. 2025 Oct 17.

ID2 suppresses multiple myeloma cell proliferation by repressing the activity of the transcription factor TCF3.

Mariateresa Fulciniti, Yao Yao, Tommaso Perini, Jessica Fong Ng, Anaïs Schavgoulidze, Shuhui Deng, Jian Cui, Jessica Encinas Mayoral, Francesco Ladisa, Ryan M Young, Charles B Epstein, Cassandra M White, Christopher J Ott, Annamaria Gulla, Shannon M Matulis, Adam S Sperling, Eugenio Morelli, Lawrence H Boise, Moritz Binder, Raphael Szalat, Mehmet K Samur, Kenneth C Anderson, Nikhil C Munshi.

Transcription factors and their cofactors are major and selective non-oncogene dependencies in multiple myeloma (MM) cells. By performing a gain-of-function perturbation screen in human MM cell lines, we identified the ID genes as putative suppressors of MM cell fitness. Among them, ID2 was found to be downregulated in MM patient cells and acted as a tumor suppressor by directly binding and repressing the basic helix loop helix factor TCF3, also known as E2A. Lower ID2 expression in MM cells conferred a proliferative advantage by increasing TCF3 activity, leading to a dependency on this transcription factor. In contrast, ID2 overexpression reduced TCF3 binding to DNA, which resulted in cell-cycle arrest and a halt in MM cell proliferation. The myeloma bone marrow milieu supported this process by further decreasing the expression of ID2 and enhancing TCF3 activity, partly via IL-6, revealing a mechanism by which the tumor microenvironment impacts MM cell behavior.

DOI: https://doi.org/10.1158/2643-3230.BCD-25-0048
Cell Death Dis. 2025 Oct 13. 16(1): 720

Intracellular pH dynamics respond to extracellular matrix stiffening and mediate vasculogenic mimicry through β-catenin.

Leah M Lund, Angelina N Marchi, Laura Alderfer, Eva Hall, Jacob Hammer, Riley Moremen, Ijeoma Asilebo, Keelan J Trull, Donny Hanjaya-Putra, Katharine A White.

Dysregulated intracellular pH (pHi) dynamics and an altered tumor microenvironment have emerged as drivers of cancer cell phenotypes. However, the molecular integration between the physical properties of the microenvironment and dynamic intracellular signaling responses remains unclear. Here, we identify a mechanistic link between extracellular matrix (ECM) stiffness and pHi dynamics in driving vasculogenic mimicry (VM), an aggressive cancer phenotype associated with poor prognosis. We performed single-cell imaging of pHi in lung and breast metastatic cell lines cultured on tunable-stiffness hydrogel systems. We used two tunable-stiffness hydrogel systems to independently model ECM stiffness induced by increased protein secretion (Matrigel) and increased protein crosslinking (Hyaluronic acid gels). We show that increased ECM stiffness lowers single-cell pHi in both lung and breast metastatic cell lines. We also observed that stiff ECM promotes a distinct morphological phenotype called vasculogenic mimicry (VM). Importantly, we show that low pHi is a necessary mediator of VM, as raising pHi on stiff ECM reduces VM phenotypes. We also find that lowering pHi on soft ECM was sufficient to induce VM in the absence of extracellular stiffening. We characterized β-catenin as a pH-dependent molecular mediator of VM, where stiffness-driven increases in β-catenin abundance can be overridden by high pHi, which destabilizes β-catenin to reduce VM on stiff ECM. In contrast, the VM-associated transcription factor FOXC2 is activated by ECM stiffness but is insensitive to pHi, and its activity alone is insufficient to maintain VM at high pHi when β-catenin is lost. We uncover a novel mechanotransduction axis in which ECM stiffness regulates intracellular pH to drive β-catenin-induced VM. We also show pHi dynamics can override mechanosensitive cell responses to the extracellular microenvironment. Thus, our work positions pHi as an integrator of mechanotransduction in cancer, suggesting a new framework for therapeutically targeting pHi in cancer and perhaps in other diseases driven by ECM remodeling.

DOI: https://doi.org/10.1038/s41419-025-08014-z
EMBO Rep. 2025 Oct 16.

Cysteine-reactive covalent chloro-N-acetamide ligands induce ferroptosis mediated cell death.

Gina Gotthardt, Janik Weckesser, Georg Tascher, Sara Barros da Gama, Hannah J Uckelmann, Shibo Sun, Martin P Schwalm, Thorsten Mosler, Giulio Ferrario, José Pedro Friedmann Angeli, Christian Münch, Stefan Knapp, Stefan Müller.

  Covalent inhibitors are an attractive targeting strategy that has expanded the development of degraders to target poorly druggable proteins including the E3 ligase RNF4. We show that RNF4 is a potential vulnerability of AML. High RNF4 expression levels correlate with poor patient survival and depletion of RNF4 results in increased sensitivity of AML cells to antileukemic drugs. Therefore, we aimed to develop chemical degraders (PROTACs) of RNF4 using a known covalent RNF4 ligand (CCW16), containing a chloro-N-acetamide group, as well as established E3 ligands targeting CRBN or VHL. However, while CCW16 and CCW16-derived PROTACs react potently with cysteines in recombinant RNF4, in cells, CCW16 forms covalent bonds with a large number of proteins, including peroxiredoxins. Consequently, CCW16 based PROTACs do not trigger degradation of RNF4, but induce the ferroptosis marker heme oxygenase-1 and impair cell viability in a distinct, RNF4-independent, ferroptotic cell death pathway. We hypothesize that other chloro-N-acetamide-containing E3 ligase ligands would also induce ferroptosis. Indeed, the RNF114 ligand EN219 also strongly induces ferroptosis, suggesting that ligands harboring this electrophile induce undesired off-target toxicity.

Keywords:  AML; CCW16; Covalent PROTACs; Ferroptosis; RNF4

DOI:  https://doi.org/10.1038/s44319-025-00593-4
Methods Mol Biol. 2026 ;2976 103-118

Mass Spectrometry-Based Proteomic Analysis of Lysosome-Enriched Fractions.

Sara Bonini, Pathma Muthukottiappan, Dominic Winter.

  Lysosomes are the main degradative organelles of most mammalian cells, playing a crucial role in nutrient metabolism and acting as a signaling hub. Due to the low abundance of lysosomes, several enrichment techniques have been developed to facilitate their investigation. Combination of these approaches with mass spectrometry-based proteomics presents a powerful tool for the unbiased characterization of the lysosomal proteome under various conditions. Different enrichment strategies result in lysosome-enriched fractions with unique characteristics, varying, for example, in protein concentration and buffer composition. Therefore, in order to obtain optimal results, the subsequent strategy for proteomics sample preparation has to be adapted. Here, we describe different methods for the processing of lysosome-enriched fractions for mass spectrometry-based proteomic analyses and provide guidance for the selection of the appropriate strategy. We further elaborate on typical parameters for mass spectrometric analysis and data processing.

Keywords:  Lysosomes; Mass spectrometry; Proteomics; RapiGest; SP3; Urea

DOI:  https://doi.org/10.1007/978-1-0716-4844-5_9
Sci Adv. 2025 Oct 17. 11(42): eadx8662

Small-molecule OPA1 inhibitors reverse mitochondrial adaptations to overcome therapy resistance in acute myeloid leukemia.

Sofia La Vecchia, Saurav Doshi, Petros Antonoglou, Tanima Kundu, Wafa Al Santli, Kleopatra Avrampou, Matthew T Witkowski, Anna Pellattiero, Federico Magrin, Kristina Ames, Amit Verma, Kira Gritsman, Xiaoyang Su, Andrea Mattarei, Iannis Aifantis, Luca Scorrano, Christina Glytsou.

Acute myeloid leukemia (AML) is the most prevalent and deadliest adult leukemia. Its frontline treatment uses the BH3 mimetic venetoclax to trigger mitochondria-dependent apoptosis. However, drug resistance nearly always develops, calling for therapies to circumvent it. Advanced microscopy and genome-wide CRISPRi screen analyses pinpointed mitochondrial adaptations primarily mediated by the master regulator of cristae shape optic atrophy 1 (OPA1) as critical for BH3 mimetics resistance. Resistant AML cells up-regulate OPA1 to modify their mitochondrial structure and evade apoptosis. MYLS22 and Opitor-0, two specific and nontoxic OPA1 inhibitors, promote apoptotic cristae remodeling and cytochrome c release, synergizing with venetoclax in AML cells and xenografts derived from AML patients ex vivo and in vivo. Mechanistically, OPA1 loss renders AML cells dependent on glutamine and sensitizes them to ferroptosis by activating ATF4-regulated integrated stress responses. Overall, our data clarify how OPA1 up-regulation allows AML cells' metabolic flexibility and survival and nominates specific OPA1 inhibitors as efficacious tools to overcome venetoclax resistance in leukemia.

DOI: https://doi.org/10.1126/sciadv.adx8662
Sci Signal. 2025 Oct 14. 18(908): eadv1529

The E3 ubiquitin ligase ZNRF3 restricts WNT receptor complex activity by stimulating the selective degradation of WNT-engaged FZD.

Bo Lu, Feng Cong.

Ligands of the WNT family induce formation of the WNT receptor signalosome and promote stabilization of the transcriptional coactivator β-catenin. The homologous transmembrane E3 ubiquitin ligases ZNRF3 and RNF43 inhibit WNT-dependent stabilization of β-catenin by stimulating the degradation of the WNT receptor FZD, whereas the secreted R-spondin proteins promote the stabilization of FZD by inducing the degradation of ZNRF3 and RNF43. Here, we report that the R-spondin-induced stabilization of β-catenin in HEK293 cells was not mimicked by FZD overexpression, highlighting a gap in our understanding of this important regulatory mechanism. Contrary to the conventional view that ZNRF3 constitutively mediates the ubiquitylation and degradation of FZD, we found that ZNRF3-induced FZD degradation depended on endogenous WNT and that ZNRF3 selectively degraded WNT-engaged FZD. WNT enhanced the association between FZD and the intracellular adaptor protein DVL, and DVL subsequently recruited ZNRF3 to FZD to promote FZD degradation. Our data suggest that WNT signaling actively restricts itself through ZNRF3-dependent degradation of WNT-engaged FZD and that R-spondin enhances WNT signaling by prolonging the action of the WNT-engaged FZD complex, rather than by simply increasing the abundance of FZD on the cell surface.

DOI: https://doi.org/10.1126/scisignal.adv1529
J Clin Med. 2025 Oct 04. pii: 7032. [Epub ahead of print]14(19):

Viable and Functional: Long-Term -80 °C Cryopreservation Sustains CD34+ Integrity and Transplant Success.

Ibrahim Ethem Pinar, Muge Sahin, Vildan Gursoy, Tuba Ersal, Ferah Budak, Vildan Ozkocaman, Fahir Ozkalemkas.

  Background: Cryopreservation of hematopoietic stem cells (HSCs) at -80 °C using uncontrolled-rate freezing is frequently employed in resource-constrained settings, yet concerns remain regarding long-term viability and clinical efficacy. Reliable post-thaw assessment is essential to ensure graft quality and engraftment success. Methods: This single-center, retrospective study evaluated 72 cryopreserved stem cell products from 25 patients stored at -80 °C for a median of 868 days. Viability was assessed using both acridine orange (AO) staining and 7-AAD (7-aminoactinomycin D) flow cytometry at three time points: collection (T0), pre-infusion (T1), and delayed post-thaw evaluation (T2). Associations between viability loss, storage duration, and clinical engraftment outcomes were analyzed. Results: Median post-thaw viability remained high (94.8%) despite a moderate time-dependent decline (~1.02% per 100 days; R2 = 0.283, p < 0.001). Mean viability loss at T2 was 9.2% (AO) and 6.6% (flow cytometry). AO demonstrated greater sensitivity to delayed degradation, with a significant difference between methods (p < 0.001). Engraftment kinetics were preserved in most patients, with neutrophil and platelet recovery primarily influenced by disease type rather than product integrity. Notably, storage duration and donor age were not significantly associated with engraftment outcomes or CD34+ cell dose. Conclusions: Long-term cryopreservation at -80 °C maintains HSC viability sufficient for durable engraftment, despite gradual decline. While transplant outcomes are primarily dictated by disease biology and remission status, AO staining provides enhanced sensitivity for detecting delayed cellular damage. Notably, our viability-loss model offers a practical framework for predicting product quality, potentially supporting graft selection and clinical decision-making in real-world, resource-constrained transplant settings.

Keywords:  CD34+ viability; cryopreservation; engraftment; flow cytometry; hematopoietic stem cell transplantation; −80 °C storage

DOI:  https://doi.org/10.3390/jcm14197032