bims-scepro 2025-03-23 papers

bims-scepro

Biomed News

on Stem cell proteostasis

Issue of 2025–03–23
twenty-two papers selected by
William Grey, University of York

Lactate dehydrogenase A-coupled NAD+ regeneration is critical for acute myeloid leukemia cell survival.
Ex Vivo Expansion of Cord Blood Hematopoietic Stem and Progenitor Cells.
Single-cell panleukemia signatures of HSPC-like blasts predict drug response and clinical outcome.
Guanine nucleotide biosynthesis blockade impairs MLL complex formation and sensitizes leukemias to menin inhibition.
The proto-oncogenic miR-106a-363 cluster enhances adverse risk acute myeloid leukemia through mitochondrial activation.
Pbx3-mediated suppression of type I interferon response contributes to leukemia progression driven by MLL-AF9.
HemAtlas: A Multi-omics Hematopoiesis Database.
Proteostasis Disruption in Inherited Bone Marrow Failure Syndromes.
Clonal hematopoiesis is clonally unrelated to multiple myeloma and is associated with specific microenvironmental changes.
Developmental mitochondrial complex I activity determines lifespan.
Deciphering the dark cancer phosphoproteome using machine-learned co-regulation of phosphosites.
Hierarchical Lineage Tracing Reveals Diverse Pathways of AML Treatment Resistance.
High resolution profiling of cell cycle-dependent protein and phosphorylation abundance changes in non-transformed cells.
TP53 mutations and TET2 deficiency cooperate to drive leukemogenesis and establish an immunosuppressive environment.
Use of Human Acute Myeloid Leukemia Cells to Study Graft-Versus-Leukemia Immunity in Xenogeneic Mouse Models of Graft-Versus-Host Disease.
Spatial proteomics and transcriptomics characterization of tissue and multiple cancer types including decalcified marrow.
Trends in Mass Spectrometry-Based Single-Cell Proteomics.
Multi-omics integration reveals immune hallmarks and biomarkers associated with FLT3 inhibitor sensitivity in FLT3-mutated AML.
HaloPROTAC3 treatment activates the unfolded protein response of the endoplasmic reticulum in non-engineered mammalian cell lines.
Unraveling genotype-phenotype associations and predictive modeling of outcome in acute myeloid leukemia.
Loss of BCL7A Permits IRF4 Transcriptional Activity and Cellular Growth in Multiple Myeloma.
Single-Cell Proteomic Characterization of Drug-Resistant Prostate Cancer Cells Reveals Molecular Signatures Associated with Morphological Changes.

bioRxiv. 2025 Mar 07. pii: 2025.03.03.641255. [Epub ahead of print]

Lactate dehydrogenase A-coupled NAD+ regeneration is critical for acute myeloid leukemia cell survival.

Ayşegül Erdem, Séléna Kaye, Francesco Caligiore, Manuel Johanns, Fleur Leguay, Jan Jacob Schuringa, Keisuke Ito, Guido Bommer, Nick van Gastel.

Enhanced glycolysis plays a pivotal role in fueling the aberrant proliferation, survival and therapy resistance of acute myeloid leukemia (AML) cells. Here, we aimed to elucidate the extent of glycolysis dependence in AML by focusing on the role of lactate dehydrogenase A (LDHA), a key glycolytic enzyme converting pyruvate to lactate coupled with the recycling of NAD+. We compared the glycolytic activity of primary AML patient samples to protein levels of metabolic enzymes involved in central carbon metabolism including glycolysis, glutaminolysis and the tricarboxylic acid cycle. To evaluate the therapeutic potential of targeting glycolysis in AML, we treated AML primary patient samples and cell lines with pharmacological inhibitors of LDHA and monitored cell viability. Glycolytic activity and mitochondrial oxygen consumption were analyzed in AML patient samples and cell lines post-LDHA inhibition. Perturbations in global metabolite levels and redox balance upon LDHA inhibition in AML cells were determined by mass spectrometry, and ROS levels were measured by flow cytometry. Among metabolic enzymes, we found that LDHA protein levels had the strongest positive correlation with glycolysis in AML patient cells. Blocking LDHA activity resulted in a strong growth inhibition and cell death induction in AML cell lines and primary patient samples, while healthy hematopoietic stem and progenitor cells remained unaffected. Investigation of the underlying mechanisms showed that LDHA inhibition reduces glycolytic activity, lowers levels of glycolytic intermediates, decreases the cellular NAD+ pool, boosts OXPHOS activity and increases ROS levels. This increase in ROS levels was however not linked to the observed AML cell death. Instead, we found that LDHA is essential to maintain a correct NAD+/NADH ratio in AML cells. Continuous intracellular NAD+ supplementation via overexpression of water-forming NADH oxidase from Lactobacillus brevis in AML cells effectively increased viable cell counts and prevented cell death upon LDHA inhibition. Collectively, our results demonstrate that AML cells critically depend on LDHA to maintain an adequate NAD+/NADH balance in support of their abnormal glycolytic activity and biosynthetic demands, which cannot be compensated for by other cellular NAD+ recycling systems. These findings also highlight LDHA inhibition as a promising metabolic strategy to eradicate leukemic cells.

DOI: https://doi.org/10.1101/2025.03.03.641255
Methods Mol Biol. 2025 Mar 20.

Ex Vivo Expansion of Cord Blood Hematopoietic Stem and Progenitor Cells.

Pinar Yurdakul-Mesutoglu, Hasan Yalim Akin, Zeynep Gunaydin.

  Umbilical cord blood (CB)-derived hematopoietic stem and progenitor cells (HSPCs) hold immense potential for regenerative medicine, particularly in hematologic malignancies and immune disorders. CB offers several advantages, including easy collection and reduced risk of graft-versus-host disease compared to other sources, like bone marrow. However, the clinical application of CB is often limited due to the relatively small number of HSPCs present in CB grafts, which can be insufficient for adult patients. This limitation has prompted researchers to explore various methods to expand HSPCs ex vivo. As research continues to refine expansion techniques, the future of CB HSPC therapy appears increasingly promising, offering new hope for patients requiring stem cell transplantation. Approaches to HSPC expansion include the use of cytokines, small molecules, epigenetic modulators, and advanced culture systems that mimic the bone marrow niche as well as emerging techniques such as gene editing. Of the key CB HSPC expansion methodologies, the use of epigenetic modifiers is among the most promising strategies for inducing proliferation while maintaining the stemness of CB HSPC. This section summarizes key methodologies for CB HSPC expansion and their transformative impact on clinical practice while providing a validated protocol for ex vivo expansion of CB-derived HSPCs using valproic acid and/or nicotinamide.

Keywords:  Cord blood; Ex vivo expansion; Hematopoietic stem and progenitor cells; Nicotinamide; Valproic acid

DOI:  https://doi.org/10.1007/7651_2025_610
Blood. 2025 Mar 16. pii: blood.2024027270. [Epub ahead of print]

Single-cell panleukemia signatures of HSPC-like blasts predict drug response and clinical outcome.

Changya Chen, Jason Xu, Jonathan H Sussman, Tiffaney L Vincent, Joseph S Tumulty, Satoshi Yoshimura, Fatemeh Alikarami, Wenbao Yu, Yang-Yang Ding, Chia-Hui Chen, Elizabeth Y Li, Austin Yang, Xiaohuan Qin, Shovik Bandyopadhyay, Jacqueline Jiahui Peng, Petri Pölönen, Haley Newman, Brent Wood, Jianzhong Hu, Rawan Shraim, Andrew D Hughes, Caroline Diorio, Lahari Uppuluri, Gongping Shi, Theresa Ryan, Tori Fuller, Mignon L Loh, Elizabeth A Raetz, Stephen P Hunger, Stanley B Pounds, Charles G Mullighan, David Frank, Jun J Yang, Kathrin Maria Bernt, David Trent Teachey, Kai Tan.

The critical role of leukemic initiating cells as a therapy-resistant population in myeloid leukemia is well established, however, the molecular signatures of such cells in acute lymphoblastic leukemia remain underexplored. Moreover, their role in therapy response and patient prognosis is yet to be systematically investigated across various types of acute leukemia. We employed single-cell multiomics to analyze diagnostic specimens from 96 pediatric patients with acute lymphoblastic, myeloid, and lineage ambiguous leukemias. Through the integration of single-cell multiomics with extensive bulk RNA-Seq and clinical datasets, we uncovered a prevalent, chemotherapy-resistant subpopulation that resembles hematopoietic stem and progenitor cells (HSPC-like) and is associated with poor clinical outcomes across all subtypes investigated. We identified a core transcriptional regulatory network (TRN) in HSPC-like blasts that is combinatorically controlled by HOXA/AP1/CEBPA. This TRN signature can predict chemotherapy response and long-term clinical outcomes. We identified shared potential therapeutic targets against HSPC-like blasts, including FLT3, BCL2, and the PI3K pathway. Our study provides a framework for linking intra-tumoral heterogeneity with therapy response, patient outcome and discovery of new therapeutic targets for pediatric acute leukemias.

DOI: https://doi.org/10.1182/blood.2024027270
Nat Commun. 2025 Mar 18. 16(1): 2641

Guanine nucleotide biosynthesis blockade impairs MLL complex formation and sensitizes leukemias to menin inhibition.

Xiangguo Shi, Minhua Li, Zian Liu, Jonathan Tiessen, Yuan Li, Jing Zhou, Yudan Zhu, Swetha Mahesula, Qing Ding, Lin Tan, Mengdie Feng, Yuki Kageyama, Yusuke Hara, Jacob J Tao, Xuan Luo, Kathryn A Patras, Philip L Lorenzi, Suming Huang, Alexandra M Stevens, Koichi Takahashi, Ghayas C Issa, Md Abul Hassan Samee, Michalis Agathocleous, Daisuke Nakada.

Targeting the dependency of MLL-rearranged (MLLr) leukemias on menin with small molecule inhibitors has opened new therapeutic strategies for these poor-prognosis diseases. However, the rapid development of menin inhibitor resistance calls for combinatory strategies to improve responses and prevent resistance. Here we show that leukemia stem cells (LSCs) of MLLr acute myeloid leukemia (AML) exhibit enhanced guanine nucleotide biosynthesis, the inhibition of which leads to myeloid differentiation and sensitization to menin inhibitors. Mechanistically, targeting inosine monophosphate dehydrogenase 2 (IMPDH2) reduces guanine nucleotides and rRNA transcription, leading to reduced protein expression of LEDGF and menin. Consequently, the formation and chromatin binding of the MLL-fusion complex is impaired, reducing the expression of MLL target genes. Inhibition of guanine nucleotide biosynthesis or rRNA transcription further suppresses MLLr AML when combined with a menin inhibitor. Our findings underscore the requirement of guanine nucleotide biosynthesis in maintaining the function of the LEDGF/menin/MLL-fusion complex and provide a rationale to target guanine nucleotide biosynthesis to sensitize MLLr leukemias to menin inhibitors.

DOI: https://doi.org/10.1038/s41467-025-57544-9
Leukemia. 2025 Mar 17.

The proto-oncogenic miR-106a-363 cluster enhances adverse risk acute myeloid leukemia through mitochondrial activation.

Nadine Sperb, Irina A Maksakova, Leo Escano, Libin Abraham, Liam MacPhee, Ariene Cabantog, Dexter Kim, Mansen Yu, Kathrin Krowiorz, Junbum Im, Sarah Grasedieck, Nicole Pochert, Christoph Ruess, Reinhild Rösler, Stephane Flibotte, Tobias Maetzig, Enrico Calzia, Lars Palmqvist, Sebastian Wiese, Linda Fogelstrand, Michael R Gold, Arefeh Rouhi, Florian Kuchenbauer.

We investigated the clinical and functional role of the miR-106a-363 cluster in adult acute myeloid leukemia (AML). LAML miRNA-Seq TCGA analyses revealed that high expression of miR-106a-363 cluster members was associated with inferior survival, and miR-106a-5p and miR-20b-5p levels were significantly elevated in patients with adverse risk AML. Overexpression of the miR-106a-363 cluster and its individual members in a murine AML model significantly accelerated leukemogenesis. Proteomics analysis of leukemic bone marrow cells from these models emphasized the deregulation of proteins involved in intracellular transport, protein complex organization and mitochondrial function, driven predominantly by miR-106a-5p. These molecular alterations suggested mitochondrial activation as a potential mechanism for the observed increase in leukemogenicity. High-resolution respirometry and STED microscopy confirmed that miR-106a-5p enhances mitochondrial respiratory activity and increases mitochondrial volume. These findings demonstrate that the miR-106a-363 cluster, and particularly miR-106a-5p, contribute to AML progression through modulation of mitochondrial function and deregulation of mitochondria-coordinated pathways.

DOI: https://doi.org/10.1038/s41375-025-02558-x
Cancer Gene Ther. 2025 Mar 19.

Pbx3-mediated suppression of type I interferon response contributes to leukemia progression driven by MLL-AF9.

Li Tang, Meng Lu, Yulong Du, Jianlong Sun.

Cell-intrinsic repression of inflammatory signaling supports the survival of acute myeloid leukemia blasts. However, how the cell-intrinsic inflammation status changes during AML progression remains elusive. Here, we used CRISPR-mediated genome editing to create a murine AML model driven by a chromosomal translocation between the mixed-lineage leukemia (Mll) gene and the Mllt3/Af9 gene. The resulting MLL-AF9 (MA9) fusion protein is sufficient to immortalize hematopoietic stem and progenitor cells (HSPCs) in vitro but insufficient to induce an overt leukemia phenotype in vivo rapidly. Leukemia progression in vivo is associated with a downregulation of type I interferon response genes, and this process depends on the upregulation of MA9 transcriptional target Pbx3 in the progenitor cell compartment. Accordingly, enhancing interferon response by interferon-α (IFNα) administration induces leukemic cell differentiation, and inhibiting MA9 transcriptional activity on top of the enhanced IFN signaling further delays leukemia progression. Our study underscores the importance of Pbx3-mediated suppression of interferon response genes in the progression of MA9-induced AML and highlights the potential application of type I interferon for its treatment.

DOI: https://doi.org/10.1038/s41417-025-00888-7
Genomics Proteomics Bioinformatics. 2025 Mar 19. pii: qzaf026. [Epub ahead of print]

HemAtlas: A Multi-omics Hematopoiesis Database.

Zhixin Kang, Tongtong Zhu, Dong Zou, Mengyao Liu, Yifan Zhang, Lu Wang, Zhang Zhang, Feng Liu.

  Advancements in high-throughput omics technologies have facilitated a systematic exploration of crucial hematopoietic organs across diverse species. A thorough understanding of hematopoiesis in vivo and facilitation of generating functional hematopoietic stem and progenitor cells (HSPCs) in vitro necessitate a comprehensive hematopoietic cross-stage developmental landscape across species. To address this need, we developed HemAtlas, a platform designed for the systematic mapping of hematopoiesis both in vivo and in vitro. HemAtlas features detailed analyses of multi-omics datasets from humans, mice, zebrafish, and HSPC in vitro culture systems. Utilizing literature curation and data normalization, HemAtlas integrates various functional modules, allowing interactive exploration and visualization of any collected omics data based on user-specific interests. Moreover, by applying a systematic and uniform integration method, we constructed organ-wide hematopoietic references for each species with manually curated cell annotations, enabling a comprehensive decoding of cross-stage developmental hematopoiesis at the organ level. Of particular significance are three distinctive functions-single-cell cross-stage, cross-species, and cross-model analysis-that HemAtlas employs to elucidate the hematopoietic development in zebrafish, mice, and humans, and to offer guidance on the generation of HSPCs in vitro. Simultaneously, HemAtlas incorporates a comprehensive map of HSPC cross-stage development to reveal HSPC stage-specific properties. Taken together, HemAtlas serves as a crucial resource to advance our understanding of hematopoiesis and is available at https://ngdc.cncb.ac.cn/hematlas/.

Keywords:  Cross-species analyses; Developmental hematopoiesis; Hematopoietic database; Hematopoietic stem and progenitor cells in vitro; Multi-omics

DOI:  https://doi.org/10.1093/gpbjnl/qzaf026
Blood. 2025 Mar 16. pii: blood.2024024956. [Epub ahead of print]

Proteostasis Disruption in Inherited Bone Marrow Failure Syndromes.

Helena Yu, Robert Signer.

Inherited bone marrow failure syndromes (IBMFS) are genetic disorders of impaired hematopoiesis that manifest in childhood with both cytopenias and extra-hematologic findings. While several IBMFS are categorized as ribosomopathies due to shared underlying ribosomal dysfunction, there is a broader disruption of the protein homeostasis (proteostasis) network across both classic and emerging IBMFS. Precise regulation of the proteostasis network, including mechanisms of protein synthesis, folding, trafficking, and degradation as well as associated stress response pathways, has emerged as essential for maintaining hematopoietic stem cell (HSC) function, providing new potential mechanistic insights into IBMFS pathogenesis. Furthermore, the varied clinical trajectories of patients with IBMFS with possible divergent outcomes of malignancy and spontaneous remission may reflect developmental and temporal changes in proteostasis activity and be driven by strong selective pressures to restore proteostasis. These new insights are spurring fresh therapeutic approaches to target proteostasis. Thus, further evaluation of proteostasis regulation and the consequences of proteostasis disruption in IBMFS could aid in developing new biomarkers, therapeutic agents, and preventative approaches for patients.

DOI: https://doi.org/10.1182/blood.2024024956
Blood. 2025 Mar 20. pii: blood.2024026236. [Epub ahead of print]

Clonal hematopoiesis is clonally unrelated to multiple myeloma and is associated with specific microenvironmental changes.

Marta Lionetti, Margherita Scopetti, Antonio Matera, Akihiro Maeda, Alessio Marella, Francesca Lazzaroni, Giancarlo Castellano, Sonia Fabris, Stefania Pioggia, Silvia Lonati, Alfredo Marchetti, Alessandra Cattaneo, Marta Tornese, Antonino Neri, Claudia Leoni, Loredana Pettine, Valentina Traini, Ilaria Silvestris, Marzia Barbieri, Giuseppina Fabbiano, Domenica Ronchetti, Elisa Taiana, Claudio De Magistris, Matteo Claudio Da Via', Francesco Passamonti, Niccolò Bolli.

Multiple myeloma (MM) initiation is dictated by genomic events. However, its progression from asymptomatic stages to an aggressive disease that ultimately fails to respond to treatments is also dependent on changes of the tumor microenvironment (TME). Clonal hematopoiesis of indeterminate potential (CHIP) is a prevalent clonal condition of the hematopoietic stem cell whose presence is causally linked to a more inflamed microenvironment. Here, we show in 106 patients with MM that CHIP is frequently co-existing with MM at diagnosis, associates with a more advanced R-ISS stage, higher age and shows a non-significant trend towards lower median hemoglobin. In our cohort the two conditions do not share a clonal origin. Single cell RNA-sequencing in 16 MM patients highlights significant TME changes when CHIP is present: decreased naïve T cells, a pro-inflammatory TME, decreased antigen-presenting function by dendritic cells and expression of exhaustion markers in CD8 cells. Inferred interactions between cell types in CHIP-positive TME suggested that especially monocytes, T cells and clonal plasma cells may have a prominent role in mediating inflammation, immune evasion and pro-survival signals in favor of MM cells. Altogether, our data show that, in the presence of CHIP, the TME of MM at diagnosis is significantly disrupted in line with what usually seen in more advanced disease, with potential translational implications. Our data highlight the relevance of this association and prompt for further studies on the modifier role of CHIP in the MM TME.

DOI: https://doi.org/10.1182/blood.2024026236
EMBO Rep. 2025 Mar 17.

Developmental mitochondrial complex I activity determines lifespan.

Rhoda Stefanatos, Fiona Robertson, Beatriz Castejon-Vega, Yizhou Yu, Alejandro Huerta Uribe, Kevin Myers, Tetsushi Kataura, Viktor I Korolchuk, Oliver D K Maddocks, L Miguel Martins, Alberto Sanz.

  Aberrant mitochondrial function has been associated with an increasingly large number of human disease states. Observations from in vivo models where mitochondrial function is altered suggest that maladaptations to mitochondrial dysfunction may underpin disease pathology. We hypothesized that the severity of this maladaptation could be shaped by the plasticity of the system when mitochondrial dysfunction manifests. To investigate this, we have used inducible fly models of mitochondrial complex I (CI) dysfunction to reduce mitochondrial function at two stages of the fly lifecycle, from early development and adult eclosion. Here, we show that in early life (developmental) mitochondrial dysfunction results in severe reductions in survival and stress resistance in adulthood, while flies where mitochondrial function is perturbed from adulthood, are long-lived and stress resistant despite having up to a 75% reduction in CI activity. After excluding developmental defects as a cause, we went on to molecularly characterize these two populations of mitochondrially compromised flies, short- and long-lived. We find that our short-lived flies have unique transcriptomic, proteomic and metabolomic responses, which overlap significantly in discrete models of CI dysfunction. Our data demonstrate that early mitochondrial dysfunction via CI depletion elicits a maladaptive response, which severely reduces survival, while CI depletion from adulthood is insufficient to reduce survival and stress resistance.

Keywords:  Ageing; Complex I; Drosophila; Mitochondria; Mitochondrial Disease

DOI:  https://doi.org/10.1038/s44319-025-00416-6
Nat Commun. 2025 Mar 20. 16(1): 2766

Deciphering the dark cancer phosphoproteome using machine-learned co-regulation of phosphosites.

Wen Jiang, Eric J Jaehnig, Yuxing Liao, Zhiao Shi, Tomer M Yaron-Barir, Jared L Johnson, Lewis C Cantley, Bing Zhang.

Mass spectrometry-based phosphoproteomics offers a comprehensive view of protein phosphorylation, yet our limited knowledge about the regulation and function of most phosphosites hampers the extraction of meaningful biological insights. To address this challenge, we integrate machine learning with phosphoproteomic data from 1195 tumor specimens spanning 11 cancer types to construct CoPheeMap, a network that maps the co-regulation of 26,280 phosphosites. By incorporating network features from CoPheeMap into a second machine learning model, namely CoPheeKSA, we achieve superior performance in predicting kinase-substrate associations. CoPheeKSA uncovers 24,015 associations between 9399 phosphosites and 104 serine/threonine kinases, shedding light on many unannotated phosphosites and understudied kinases. We validate the accuracy of these predictions using experimentally determined kinase-substrate specificities. Through the application of CoPheeMap and CoPheeKSA to phosphosites with high computationally predicted functional significance and those associated with cancer, we demonstrate their effectiveness in systematically elucidating phosphosites of interest. These analyses unveil dysregulated signaling processes in human cancer and identify understudied kinases as potential therapeutic targets.

DOI: https://doi.org/10.1038/s41467-025-57993-2
bioRxiv. 2025 Mar 03. pii: 2025.02.27.640600. [Epub ahead of print]

Hierarchical Lineage Tracing Reveals Diverse Pathways of AML Treatment Resistance.

Rachel Saxe, Hannah Stuart, Abigail Marshall, Fahiima Abdullahi, Zoë Chen, Francesco Emiliani, Aaron McKenna.

Cancer cells adapt to treatment, leading to the emergence of clones that are more aggressive and resistant to anti-cancer therapies. We have a limited understanding of the development of treatment resistance as we lack technologies to map the evolution of cancer under the selective pressure of treatment. To address this, we developed a hierarchical, dynamic lineage tracing method called FLARE (Following Lineage Adaptation and Resistance Evolution). We use this technique to track the progression of acute myeloid leukemia (AML) cell lines through exposure to Cytarabine (AraC), a front-line treatment in AML, in vitro and in vivo. We map distinct cellular lineages in murine and human AML cell lines predisposed to AraC persistence and/or resistance via the upregulation of cell adhesion and motility pathways. Additionally, we highlight the heritable expression of immunoproteasome 11S regulatory cap subunits as a potential mechanism aiding AML cell survival, proliferation, and immune escape in vivo. Finally, we validate the clinical relevance of these signatures in the TARGET-AML cohort, with a bisected response in blood and bone marrow. Our findings reveal a broad spectrum of resistance signatures attributed to significant cell transcriptional changes. To our knowledge, this is the first application of dynamic lineage tracing to unravel treatment response and resistance in cancer, and we expect FLARE to be a valuable tool in dissecting the evolution of resistance in a wide range of tumor types.

DOI: https://doi.org/10.1101/2025.02.27.640600
Nat Commun. 2025 Mar 16. 16(1): 2579

High resolution profiling of cell cycle-dependent protein and phosphorylation abundance changes in non-transformed cells.

Camilla Rega, Ifigenia Tsitsa, Theodoros I Roumeliotis, Izabella Krystkowiak, Maria Portillo, Lu Yu, Julia Vorhauser, Jonathon Pines, Jörg Mansfeld, Jyoti Choudhary, Norman E Davey.

The cell cycle governs a precise series of molecular events, regulated by coordinated changes in protein and phosphorylation abundance, that culminates in the generation of two daughter cells. Here, we present a proteomic and phosphoproteomic analysis of the human cell cycle in hTERT-RPE-1 cells using deep quantitative mass spectrometry by isobaric labelling. By analysing non-transformed cells and improving the temporal resolution and coverage of key cell cycle regulators, we present a dataset of cell cycle-dependent protein and phosphorylation site oscillation that offers a foundational reference for investigating cell cycle regulation. These data reveal regulatory intricacies including proteins and phosphorylation sites exhibiting cell cycle-dependent oscillation, and proteins targeted for degradation during mitotic exit. Integrated with complementary resources, our data link cycle-dependent abundance dynamics to functional changes and are accessible through the Cell Cycle database (CCdb), an interactive web-based resource for the cell cycle community.

DOI: https://doi.org/10.1038/s41467-025-57537-8
J Clin Invest. 2025 Mar 20. pii: e184021. [Epub ahead of print]

TP53 mutations and TET2 deficiency cooperate to drive leukemogenesis and establish an immunosuppressive environment.

Pu Zhang, Ethan C Whipp, Sarah J Skuli, Mehdi Gharghabi, Caner Saygin, Steven A Sher, Martin Carroll, Xiangyu Pan, Eric D Eisenmann, Tzung-Huei Lai, Bonnie K Harrington, Wing Keung Chan, Youssef Youssef, Bingyi Chen, Alex Penson, Alexander M Lewis, Cynthia R Castro, Nina Fox, Ali Cihan, Jean-Benoit Le Luduec, Susan DeWolf, Tierney Kauffman, Alice S Mims, Daniel Canfield, Hannah Phillips, Katie E Williams, Jami Shaffer, Arletta Lozanski, Tzyy-Jye Doong, Gerard Lozanski, Charlene Mao, Christopher J Walker, James S Blachly, Anthony F Daniyan, Lapo Alinari, Robert A Baiocchi, Yiping Yang, Nicole R Grieselhuber, Moray J Campbell, Sharyn D Baker, Bradley W Blaser, Omar Abdel-Wahab, Rosa Lapalombella.

  Mutations and deletions in TP53 are associated with adverse outcomes in patients with myeloid malignancies and developing improved therapies for TP53-mutant leukemias is of urgent need. Here we identify mutations in TET2 as the most common co-occurring mutation in TP53 mutant acute myeloid leukemia (AML) patients. In mice, combined hematopoietic-specific deletion of TET2 and TP53 resulted in enhanced self-renewal compared to deletion of either gene alone. Tp53/Tet2 double knockout mice developed serially transplantable AML. Both mice and AML patients with combined TET2/TP53 alterations upregulated innate immune signaling in malignant granulocyte-monocyte progenitors (GMPs), which had leukemia-initiating capacity. A20 governs the leukemic maintenance by triggering aberrant non-canonical NF-κB signaling. Mice with Tp53/Tet2 loss had expansion of monocytic myeloid-derived suppressor cells (MDSCs), which impaired T cell proliferation and activation. Moreover, mice and AML patients with combined TP53/TET2 alterations displayed increased expression of the TIGIT ligand, CD155, on malignant cells. TIGIT blocking antibodies augmented NK cell-mediated killing of Tp53/Tet2 double-mutant AML cells, reduced leukemic burden, and prolonged survival in Tp53/Tet2 double knockout mice. These findings uncover a leukemia-promoting link between TET2 and TP53 mutations and highlight therapeutic strategies to overcome the immunosuppressive bone marrow environment in this adverse subtype of AML.

Keywords:  Hematology; Inflammation; Leukemias; Mouse models; Oncology; p53

DOI:  https://doi.org/10.1172/JCI184021
Methods Mol Biol. 2025 ;2907 359-375

Use of Human Acute Myeloid Leukemia Cells to Study Graft-Versus-Leukemia Immunity in Xenogeneic Mouse Models of Graft-Versus-Host Disease.

Charline Faville, Bianca E Silva, Frédéric Baron, Grégory Ehx.

  Allogeneic hematopoietic cell transplantation (allo-HCT) is the main therapeutic approach for patients with high-risk acute myeloid leukemia (AML), but the rate of relapse remains high and is associated with poor outcomes. Discovering new approaches to maximize the graft-versus-leukemia (GVL) effects while mitigating graft-versus-host disease (GVHD) should therefore be pursued. Because of the difficulties in modeling AML in mice, patient-derived xenotransplantations (PDXs) in immunodeficient NOD-scid-IL2rgnull (NSG) mice are preferred to study the GVL effects. In PDX, AML is typically induced through the intravenous injection of cell lines or leukemic blasts obtained from patients. GVHD and GVL effects are induced by (co)-injecting human T cells or peripheral blood mononuclear cells (PBMCs). While this approach enables the induction of systemic leukemia, notably developing in the spleen and bone marrow of the animals, it can also be associated with difficulties in monitoring the disease, notably by flow cytometry. This can be circumvented by using luciferase-expressing AML cells or transplanting the leukemic cells in Matrigel to generate solid tumors that are easier to monitor. Here, we provide detailed instructions on how to prepare human PBMCs and leukemic cells, transplant them, and monitor the disease in NSG mice.

Keywords:  AML; Bioluminescence; Graft-versus-host disease; Graft-versus-leukemia effects; Hematopoietic cell transplantation; Immunodeficient mice; Xenotransplantation

DOI:  https://doi.org/10.1007/978-1-0716-4430-0_17
Cancer Biomark. 2025 Jan;42(1): 18758592241308757

Spatial proteomics and transcriptomics characterization of tissue and multiple cancer types including decalcified marrow.

Cecilia Cs Yeung, Daniel C Jones, David W Woolston, Brandon Seaton, Elizabeth Lawless Donato, Minggang Lin, Coral Backman, Vivian Oehler, Kristin L Robinson, Kristen Shimp, Rima Kulikauskas, Annalyssa N Long, David Sowerby, Anna E Elz, Kimberly S Smythe, Evan W Newell.

  BackgroundRecent technologies enabling the study of spatial biology include multiple high-dimensional spatial imaging methods that have rapidly emerged with different capabilities evaluating tissues at different resolutions for different sample formats. Platforms like Xenium (10x Genomics) and PhenoCycler-Fusion (Akoya Biosciences) enable single-cell resolution analysis of gene and protein expression in archival FFPE tissue slides. However, a key limitation is the absence of systematic methods to ensure tissue quality, marker integrity, and data reproducibility.ObjectiveWe seek to optimize the technical methods for spatial work by addressing preanalytical challenges with various tissue and tumor types, including a decalcification protocol for processing FFPE bone marrow core specimens to preserve nucleic acids for effective spatial proteomics and transcriptomics. This study characterizes a multicancer tissue microarray (TMA) and a molecular- and protein-friendly decalcification protocol that supports downstream spatial biology investigations.MethodsWe developed a multi-cancer tissue microarray (TMA) and processed bone marrow core samples using a molecular- and protein-friendly decalcification protocol. PhenoCycler high-plex immunohistochemistry (IHC) generated spatial proteomics data, analyzed with QuPath and single-cell analysis. Xenium provided spatial transcriptomics data, analyzed via Xenium Explorer and custom pipelines.ResultsResults showed that PhenoCycler and Xenium platforms applied to TMA sections of tonsil and various tumor types achieved good marker concordance. Bone marrow decalcification with our optimized protocol preserved mRNA and protein markers, allowing Xenium analysis to resolve all major cell types while maintaining tissue morphology.ConclusionsWe have shared our preanalytical verification of tissues and demonstrate that both the PhenoCycler-Fusion high-plex spatial proteomics and Xenium spatial transcriptomics platforms work well on various tumor types, including marrow core biopsies decalcified using a molecular- and protein-friendly decalcificationprotocol. We also demonstrate our laboratory's methods for systematic quality assessment of the spatial proteomic and transcriptomic data from these platforms, such that either platform can provide orthogonal confirmation for the other.

Keywords:  high-plex immunohistochemistry; multi-cancer; single cell spatial profiling; spatial biology; spatial proteomics; spatial transcriptomics; tissue microarray

DOI:  https://doi.org/10.1177/18758592241308757
Anal Chem. 2025 Mar 16.

Trends in Mass Spectrometry-Based Single-Cell Proteomics.

Ximena Sanchez-Avila, Raphaela M de Oliveira, Siqi Huang, Chao Wang, Ryan T Kelly.

DOI: https://doi.org/10.1021/acs.analchem.5c00661
Blood Sci. 2025 Jun;7(2): e00227

Multi-omics integration reveals immune hallmarks and biomarkers associated with FLT3 inhibitor sensitivity in FLT3-mutated AML.

Mingming Niu, Ning Wang, Dandan Yang, Lixia Fu, Yang Yang, Long Shen, Hong Wang, Xianfeng Shao.

  Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by poor clinical outcomes, frequently exacerbated by mutations in the FMS-like tyrosine kinase 3 (FLT3) gene. Although FLT3 inhibitors (FLT3i) have emerged as promising therapeutic agents, the absence of molecular biomarkers to predict FLT3i response remains a critical limitation in clinical practice. In this study, we performed a comprehensive multi-omics analysis integrating transcriptomic, proteomic, and pharmacogenomic data from the Beat AML cohort, the Cancer Cell Line Encyclopedia (CCLE), and the PXD023201 repository to elucidate the molecular consequences of FLT3 mutations in AML. Our analysis revealed significant differences in RNA and protein expression profiles between FLT3-mutant and wild-type AML cases, with a particularly striking association between FLT3 mutations and immune suppression. We further evaluated the drug sensitivity of FLT3-mutant patients to 3 FDA-approved FLT3i, gilteritinib, midostaurin, and quizartinib, and observed heightened sensitivity in FLT3-mutant cohorts, accompanied by the activation of immune-related pathways in treatment-responsive groups. These findings suggest a potential synergy between FLT3i efficacy and immune activation. Through rigorous bioinformatic analysis, we identified 3 candidate biomarkers: CD36, SASH1, and NIBAN2, associated with FLT3i sensitivity. These biomarkers were consistently upregulated in favorable prognostic subgroups and demonstrated strong correlations with immune activation pathways. The identification of CD36, SASH1, and NIBAN2 as predictive biomarkers offers a novel toolset for stratifying FLT3i response and prognosis.

Keywords:  Acute myeloid leukemia; Drug sensitivity; FLT3 inhibitors; FLT3 mutations; Immunity; Multi-omics

DOI:  https://doi.org/10.1097/BS9.0000000000000227
Mol Biol Cell. 2025 Mar 19. mbcE24080342

HaloPROTAC3 treatment activates the unfolded protein response of the endoplasmic reticulum in non-engineered mammalian cell lines.

Aleksandra S Anisimova, G Elif Karagöz.

Proteins fused to HaloTag, an engineered haloalkane dehalogenase, can be depleted by a heterobifunctional degrader compound HaloPROTAC3. The binding of HaloPROTAC3 to both the HaloTag and the E3 ligase von Hippel-Lindau (VHL) brings them into proximity and mediates the degradation of the HaloTag fusion proteins. Here, we generated a colon cancer cell line HCT116 expressing HaloTag fused to the RNA-binding protein IGF2BP3 to study its function. HaloPROTAC3 treatment depleted 75% of HaloTag-IGF2BP3 in 5 hours. Transcriptomics revealed that HaloPROTAC3 treatment resulted in the destabilization of IGF2BP3 target mRNAs and activated the unfolded protein response (UPR). Surprisingly, we found that HaloPROTAC3 results in UPR activation in non-engineered mammalian cells. Our data demonstrate that HaloPROTAC3 causes mild endoplasmic reticulum stress independent of IGF2BP3 function and shall guide future studies using the HaloPROTAC3 protein depletion strategy.

DOI: https://doi.org/10.1091/mbc.E24-08-0342
Cytometry B Clin Cytom. 2025 Mar 20.

Unraveling genotype-phenotype associations and predictive modeling of outcome in acute myeloid leukemia.

Artuur Couckuyt, Sofie Van Gassen, Annelies Emmaneel, Vince Janda, Malicorne Buysse, Ine Moors, Jan Philippé, Mattias Hofmans, Tessa Kerre, Yvan Saeys, Sarah Bonte.

  Acute myeloid leukemia (AML) comprises 32% of adult leukemia cases, with a 5-year survival rate of only 20-30%. Here, the immunophenotypic landscape of this heterogeneous malignancy is explored in a single-center cohort using a novel quantitative computational pipeline. For 122 patients who underwent induction treatment with intensive chemotherapy, leukemic cells were identified at diagnosis, computationally preprocessed, and quantitatively subtyped. Computational analysis provided a broad characterization of inter- and intra-patient heterogeneity, which would have been harder to achieve with manual bivariate gating. Statistical testing discovered associations between CD34, CD117, and HLA-DR expression patterns and genetic abnormalities. We found the presence of CD34+ cell populations at diagnosis to be associated with a shorter time to relapse. Moreover, CD34- CD117+ cell populations were associated with a longer time to AML-related mortality. Machine learning (ML) models were developed to predict 2-year survival, European LeukemiaNet (ELN) risk category, and inv(16) or NPM1mut, based on computationally quantified leukemic cell populations and limited clinical data, both readily available at diagnosis. We used explainable artificial intelligence (AI) to identify the key clinical characteristics and leukemic cell populations important for our ML models when making these predictions. Our findings highlight the importance of developing objective computational pipelines integrating immunophenotypic and genetic information in the risk stratification of AML.

Keywords:  acute myeloid leukemia (AML); bioinformatics; computational cytometry; computational pipeline; explainable artificial intelligence; flow cytometry; machine learning models

DOI:  https://doi.org/10.1002/cyto.b.22230
Blood. 2025 Mar 16. pii: blood.2024026588. [Epub ahead of print]

Loss of BCL7A Permits IRF4 Transcriptional Activity and Cellular Growth in Multiple Myeloma.

Chandraditya Chakraborty, Srikanth Talluri, Moritz Binder, Eugenio Morelli, Jessica Encinas Mayoral, Sanika Derebail, Anil Aktas Samur, Charles B Epstein, Kenneth C Anderson, Masood A Shammas, Mehmet K Samur, Mariateresa Fulciniti, Nikhil C Munshi.

Multiple myeloma (MM) is a complex hematological malignancy characterized by genomic changes and transcriptomic dysregulation. Initial exome sequencing approaches have failed to identify any single frequent (>25%) mutation in the coding genome. However, using whole-genome sequencing (WGS), we found that one of the genomic regions most frequently mutated (62% of the MM patients) was the 5' untranslated (UTR) region and/or intron 1 of the BCL7A gene. RNA-seq data from a large cohort suggests a loss of BCL7A expression in a large majority of MM patients as compared to normal plasma cells. BCL7A loss of function in a panel of MM cell lines led to a highly proliferative phenotype in vitro and in vivo, while its ectopic expression significantly reduced cell viability, suggesting a tumor suppressor function for BCL7A in MM. We studied the cellular and molecular effects of BCL7A loss and observed that it endows myeloma cells with proliferative potential in cooperation with the plasma cell-defining transcription factor IRF4. BCL7A is involved in a direct protein-protein interaction with IRF4, limiting its DNA binding activity. Loss of BCL7A thus enhances the expression of IRF4-associated cytokines and reduces mitochondrial metabolism and ROS levels. Our study therefore suggests that BCL7A loss provides the necessary molecular change to allow IRF4-mediated transcriptional activity and MM cell growth and survival.

DOI: https://doi.org/10.1182/blood.2024026588
Mol Cell Proteomics. 2025 Mar 14. pii: S1535-9476(25)00047-7. [Epub ahead of print] 100949

Single-Cell Proteomic Characterization of Drug-Resistant Prostate Cancer Cells Reveals Molecular Signatures Associated with Morphological Changes.

Jongmin Woo, Michael Loycano, Md Amanullah, Jiang Qian, Sarah R Amend, Kenneth J Pienta, Hui Zhang.

  This study delves into the proteomic intricacies of drug-resistant cells (DRCs) within prostate cancer, which are known for their pivotal roles in therapeutic resistance, relapse, and metastasis. Utilizing single-cell proteomics (SCP) with an optimized high-throughput Data Independent Acquisition (DIA) approach with the throughput of 60 sample per day, we characterized the proteomic landscape of DRCs in comparison to parental PC3 cells. This DIA method allowed for robust and reproducible protein quantification at the single-cell level, enabling the identification and quantification of over 1,300 proteins per cell on average. Distinct proteomic sub-clusters within the DRC population were identified, closely linked to variations in cell size. The study uncovered novel protein signatures, including the regulation of proteins critical for cell adhesion and metabolic processes, as well as the upregulation of surface proteins and transcription factors pivotal for cancer progression. Furthermore, by conducting single-cell RNA-seq (scRNA-seq) analysis, we identified six upregulated and ten downregulated genes consistently altered in drug-treated cells across both SCP and scRNA-seq platforms. These findings underscore the heterogeneity of DRCs and their unique molecular signatures, providing valuable insights into their biological behavior and potential therapeutic targets.

Keywords:  Drug-resistant; Molecular signature; Prostate cancer cell line; Single-cell Proteomics

DOI:  https://doi.org/10.1016/j.mcpro.2025.100949