bims-scepro 2024-11-17 papers

bims-scepro

Biomed News

on Stem cell proteostasis

Issue of 2024‒11‒17
34 papers selected by
William Grey, University of York

Rapid Disease Progression of Myelodysplastic Syndrome is Reflected in Transcriptomic and Functional Abnormalities of Bone Marrow MSCs.
An updated outlook on autophagy mechanism and how it supports acute myeloid leukemia maintenance.
Post-transplant G-CSF impedes engraftment of gene-edited human hematopoietic stem cells by exacerbating p53-mediated DNA damage response.
Paediatric bone marrow mesenchymal stem cells support acute myeloid leukaemia cell survival and enhance chemoresistance via contact-independent mechanism.
A CADASIL NOTCH3 mutation leads to clonal hematopoiesis and expansion of Dnmt3a-R878H hematopoietic clones.
Bone morphogenetic protein 4 induces hematopoietic stem cell development from murine hemogenic endothelial cells in culture.
Overexpression of Pin1 regulated by TOP2A, which subsequently stabilizes Pyk2 to promote bortezomib resistance in multiple myeloma.
Regulated GATA1 expression as a universal gene therapy for Diamond-Blackfan anemia.
IL-21/IL-21R signaling renders acute myeloid leukemia stem cells more susceptible to cytarabine treatment and CAR T cell therapy.
CXCL8 secreted by immature granulocytes inhibits WT hematopoiesis in chronic myelomonocytic leukemia.
Adult skull bone marrow is an expanding and resilient haematopoietic reservoir.
Mass spectrometry-based proteomic exploration of diverse murine macrophage cellular models.
Succinate dehydrogenase deficiency-driven succinate accumulation induces drug resistance in acute myeloid leukemia via ubiquitin-cullin regulation.
PU.1 eviction at lymphocyte-specific chromatin domains mediates glucocorticoid response in acute lymphoblastic leukemia.
GPR68 supports AML cells through the calcium/calcineurin pro-survival pathway and confers chemoresistance by mediating glucose metabolic symbiosis.
A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells.
Interleukin-1β induces trained innate immunity in human hematopoietic progenitor cells in vitro.
Redistribution of PU.1 partner transcription factor RUNX1 binding secures cell survival during leukemogenesis.
Molecular characterization of human HSPCs with different cell fates in vivo using single-cell transcriptome analysis and lentiviral barcoding technology.
Structural response of microtubule and actin cytoskeletons to direct intracellular load.
Parallel phosphoproteomics and metabolomics map the global metabolic tyrosine phosphoproteome.
A single-cell transcriptomic map of the murine and human multiple myeloma immune microenvironment across disease stages.
Redox Regulation of Proteostasis.
Modeling Drug Responses and Evolutionary Dynamics using Patient-Derived Xenografts Reveals Precision Medicine Strategies for Triple Negative Breast Cancer.
VPS4A is the selective receptor for lipophagy in mice and humans.
HSF-1 promotes longevity through ubiquilin-1-dependent mitochondrial network remodelling.
Preclinical evaluation of the CD38-targeting engineered toxin body MT-0169 against multiple myeloma.
Improved identification of clinically relevant Acute Leukemia subtypes using standardized EuroFlow panels versus non-standardized approach.
Force-transducing molecular ensembles at growing microtubule tips control mitotic spindle size.
α-tubulin detyrosination fine-tunes kinetochore-microtubule attachments.
Non-cell autonomous regulation of cell-cell signaling and differentiation by mitochondrial ROS.
HURP regulates Kif18A recruitment and activity to synergistically control microtubule dynamics.
Engineered receptors for soluble cellular communication and disease sensing.
A glutamine metabolic switch supports erythropoiesis.

Stem Cells. 2024 Nov 14. pii: sxae073. [Epub ahead of print]

Rapid Disease Progression of Myelodysplastic Syndrome is Reflected in Transcriptomic and Functional Abnormalities of Bone Marrow MSCs.

Hein Than, Xiubo Fan, Alice M S Cheung, William Y K Hwang, Zhiyong Poon.

  Bone marrow (BM) mesenchymal stromal cells (MSCs) are important regulators of hematopoietic stem and progenitor cells (HSPCs). When transformed to a dysplastic phenotype, MSCs contribute to hematopoietic diseases such as myelodysplastic syndromes (MDS), but it remains unclear if there are specific properties in MDS-MSCs that contribute to the disease course. To understand this, we investigated MDS-MSCs from fast (MDSfast) vs slow (MDSslow) progressing disease groups and discovered differences between these groups. MDSfast-MSCs secrete more inflammatory factors, support myeloid-skewed differentiation of HSPCs, and importantly, show poorer response to hypomethylation as a key differentiator in GSEA analysis. When exposed to long-term in vivo stimulation with primary MDSfast-MSCs-based scaffolds, healthy donor (HD) HSPCs show elevated NF-κB expression, similar to leukemic HSPCs in MDS. Those "MDSfast-MSCs-primed" HD-HSPCs continue to show enhanced engraftment rates in secondary MDS-MSC-based scaffolds, providing evidence for the microenvironmental selection pressures in MDS towards leukemic HSPCs. Together, our data point towards a degree of co-development between MSCs and HSPCs during the progression of MDS, where changes in MDS-MSCs take place mainly at the transcriptomic and functional levels. These unique differences in MDS-MSCs can be utilized to improve disease prognostication and implement targeted therapy for unmet clinical needs.

Keywords:  Myelodysplastic syndromes; bone marrow microenvironment; mesenchymal stromal cells

DOI:  https://doi.org/10.1093/stmcls/sxae073
Biochim Biophys Acta Rev Cancer. 2024 Nov 06. pii: S0304-419X(24)00145-8. [Epub ahead of print]1879(6): 189214

An updated outlook on autophagy mechanism and how it supports acute myeloid leukemia maintenance.

Brunno Gilberto Santos de Macedo, Manuela Albuquerque de Melo, Diego Antonio Pereira-Martins, João Agostinho Machado-Neto, Fabiola Traina.

  The gradual acquisition of genetic and epigenetic disturbances bestows malignant traits upon hematopoietic stem cells, subverting them into a founder and reservoir cell for de novo acute myeloid leukemia (AML) known as leukemic stem cells (LSC). Beyond its molecular heterogeneity, AML is also characterized by rewiring biological processes to support its onset and maintenance. LSC were observed to inherently and actively trigger mitochondrial turnover through selective autophagic removal such that impairing the process led to cell differentiation at the expense of its stemness. This review provides a current take on autophagy regulation mechanisms according to the current molecular characterization of the process; describes autophagy as a drug resistance mechanism, and a pivotal mechanism whereby LSC harmonize their strong reliance on mitochondrial respiration to obtain energy, and their necessity for lower internal oxidative stress to avoid exhaustion. Therefore, targeting autophagy presents a promising strategy to promote long-term remissions in AML.

Keywords:  Acute myeloid leukemia; Autophagy; Leukemic stem cell; Mitophagy; Selective autophagy

DOI:  https://doi.org/10.1016/j.bbcan.2024.189214
Cell Stem Cell. 2024 Nov 08. pii: S1934-5909(24)00375-8. [Epub ahead of print]

Post-transplant G-CSF impedes engraftment of gene-edited human hematopoietic stem cells by exacerbating p53-mediated DNA damage response.

Daisuke Araki, Vicky Chen, Neelam Redekar, Christi Salisbury-Ruf, Yan Luo, Poching Liu, Yuesheng Li, Richard H Smith, Pradeep Dagur, Christian Combs, Andre Larochelle.

  Granulocyte-colony-stimulating factor (G-CSF) is commonly used to accelerate recovery from neutropenia following chemotherapy and autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) for malignant disorders. However, its utility after ex vivo gene therapy in human HSPCs remains unexplored. We show that administering G-CSF from day 1 to 14 post-transplant impedes engraftment of CRISPR-Cas9 gene-edited human HSPCs in murine xenograft models. G-CSF affects gene-edited HSPCs through a cell-intrinsic mechanism, causing proliferative stress and amplifying the early p53-mediated DNA damage response triggered by Cas9-mediated DNA double-strand breaks. This underscores a threshold mechanism where p53 activation must reach a critical level to impair cellular function. Transiently inhibiting p53 or delaying the initiation of G-CSF treatment to day 5 post-transplant attenuates its negative impact on gene-edited HSPCs. The potential for increased HSPC toxicity associated with post-transplant G-CSF administration in CRISPR-Cas9 autologous HSPC gene therapy warrants consideration in clinical trials.

Keywords:  CRISPR-Cas9; DNA damage response; DNA double-stranded breaks; G-CSF; apoptosis; cell-cycle arrest; gene editing; granulocyte colony stimulating factor; hematopoietic stem and progenitor cells; p53 pathway

DOI:  https://doi.org/10.1016/j.stem.2024.10.013
Br J Haematol. 2024 Nov 10.

Paediatric bone marrow mesenchymal stem cells support acute myeloid leukaemia cell survival and enhance chemoresistance via contact-independent mechanism.

Alison Laing, Ahmed Elmarghany, Arwa A Alghaith, Aya Gouma, Thomas Stevens, Alexander Winton, Jennifer Cassels, Cassie J Clarke, Claire Schwab, Christine J Harrison, Brenda Gibson, Karen Keeshan.

  Children diagnosed with acute myeloid leukaemia (paediatric AML [pAML]) have limited treatment options and relapse rates due to chemoresistance and refractory disease are over 30%. Current treatment is cytotoxic and in itself has long-lasting harsh side effects. New, less toxic treatments are needed. The bone marrow microenvironment provides chemoprotection to leukaemic cells through cell communication and interaction with mesenchymal stem cells (MSCs), but this is not well defined in pAML. Using primary patient material, we identify a cell contact-independent mechanism of MSC-mediated chemoprotection involving extrinsic soluble factors that is abrogated through inhibition of the JAK/STAT and ERK pathways.

Keywords:  AML; chemosensitivity; childhood leukaemia; mesenchymal cells

DOI:  https://doi.org/10.1111/bjh.19884
Leukemia. 2024 Nov 13.

A CADASIL NOTCH3 mutation leads to clonal hematopoiesis and expansion of Dnmt3a-R878H hematopoietic clones.

Raúl Sánchez-Lanzas, Justin Barclay, Alexandros Hardas, Foteini Kalampalika, Amanda Jiménez-Pompa, Paolo Gallipoli, Miguel Ganuza.

Clonal hematopoiesis (CH) is nearly universal in the elderly. The molecular and cellular mechanisms driving CH and the clinical consequences of carrying clonally derived mutant mature blood cells are poorly understood. We recently identified a C223Y mutation in the extracellular domain (ECD) of NOTCH3 as a putative CH driver in mice. Provocatively, germline NOTCH3 ECD mutations perturbing cysteine numbers cause Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL), a type of vascular dementia, suggesting an unexpected link between CADASIL and CH. Here, we formally demonstrated that mouse hematopoietic stem and progenitor cells (HSPCs) expressing CADASIL-related NOTCH3C455R exhibit a proliferative advantage resulting in robust cellular expansion in vivo and in vitro. Co-expression of NOTCH3C455R and Dnmt3aR878H, homologous to a frequent human CH mutation, increased the fitness of NOTCH3C455R HSPCs, demonstrating their functional cooperation. Surprisingly, the presence of NOTCH3C455R hematopoietic cells supported the expansion of Dnmt3aR878H HSPCs in a non-cell autonomous fashion in vivo, strongly suggesting that CADASIL patients and asymptomatic carriers can be highly predisposed to DNMT3AR882H-driven CH. Considering that CADASIL-related NOTCH3 mutations are more frequent in the general population than anticipated (~1 carrier in 400 people), the effect of these NOTCH3 mutations on CH development should be considered.

DOI: https://doi.org/10.1038/s41375-024-02464-8
Stem Cell Reports. 2024 Nov 03. pii: S2213-6711(24)00293-5. [Epub ahead of print]

Bone morphogenetic protein 4 induces hematopoietic stem cell development from murine hemogenic endothelial cells in culture.

Mariko Tsuruda, Saori Morino-Koga, Xueyu Zhao, Shingo Usuki, Kei-Ichiro Yasunaga, Tomomasa Yokomizo, Ryuichi Nishinakamura, Toshio Suda, Minetaro Ogawa.

  Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) during mouse embryogenesis. Understanding the signaling molecules required for HSC development is crucial for the in vitro derivation of HSCs. We previously induced HSCs from embryonic HECs, isolated at embryonic day 10.5 (E10.5), in serum-free culture conditions with stem cell factor, thrombopoietin, and an endothelial feeder layer. Here, we aimed to elucidate signal requirements for inducing HSCs from earlier-stage HECs. Single-cell RNA sequencing (RNA-seq) analysis detected bone morphogenetic protein (BMP) signaling activation in E9.5 HECs. Adding BMP4 to the culture conditions led to the induction of HSCs from E9.5 HECs. Furthermore, isolating BMP4 receptor-expressing HECs from E9.5 embryos enriched progenitors with HSC-forming ability. This study identified BMP4 as an essential factor promoting the differentiation of early HECs into HSCs, opening up new possibilities for the in vitro derivation of HSCs.

Keywords:  bone marrow reconstitution; bone morphogenetic protein 4; endothelial cells; hematopoietic stem cells; hemogenic endothelial cells; mouse embryos; serum-free culture; single-cell RNA sequencing

DOI:  https://doi.org/10.1016/j.stemcr.2024.10.005
Cancer Gene Ther. 2024 Nov 07.

Overexpression of Pin1 regulated by TOP2A, which subsequently stabilizes Pyk2 to promote bortezomib resistance in multiple myeloma.

Honghao Zhang, Jianyu Chen, Yabo Meng, Qingyan Cen, Hao Wang, Xiangyang Ding, Kexin Ai, Yulu Yang, Yang Gao, Yingqi Qiu, Yuxing Hu, Meifang Li, Yanjie He, Yuhua Li.

Multiple myeloma (MM), a hematological malignancy of plasma cells, has remained largely incurable owing to drug resistance and disease relapse, which requires novel therapeutic targets and treatment approaches. Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) acts as an oncoprotein linked to the development of various tumors. However, the functional consequence of Pin1 overexpression in modulating MM biology has not been established. In the present study, we show that Pin1 expression is highly variable in myeloma cell lines and primary MMs and that high Pin1 expression is associated with poor survival of MM patients. Next, TOP2A is identified to be a Pin1 promoter-binding protein and CK2 activates TOP2A to promote the expression level of Pin1. Additionally, we demonstrate that Pin1 positively modulates the stability and function of Pyk2 to enhance bortezomib resistance in MM. Pin1 recognizes three phosphorylated Ser/Thr-Pro motifs in Pyk2 via its WW domain and increases the cellular levels of Pyk2 in an isomerase activity-dependent manner by inhibiting the ubiquitination and proteasomal degradation of Pyk2. Moreover, Pin1 inhibition combined with Pyk2 inhibition decreases myeloma burden both in vitro and in vivo. Altogether, our findings reveal the tumor-promoting role of Pin1 in MM and provide evidence that targeting Pin1 could be a therapeutic strategy for MM.

DOI: https://doi.org/10.1038/s41417-024-00845-w
Cell Stem Cell. 2024 Nov 08. pii: S1934-5909(24)00374-6. [Epub ahead of print]

Regulated GATA1 expression as a universal gene therapy for Diamond-Blackfan anemia.

Richard A Voit, Xiaotian Liao, Alexis Caulier, Mateusz Antoszewski, Blake Cohen, Myriam Armant, Henry Y Lu, Travis J Fleming, Elena Kamal, Lara Wahlster, Aoife M Roche, John K Everett, Angelina Petrichenko, Mei-Mei Huang, William Clarke, Kasiani C Myers, Craig Forester, Antonio Perez-Atayde, Frederic D Bushman, Danilo Pellin, Akiko Shimamura, David A Williams, Vijay G Sankaran.

  Gene therapy using hematopoietic stem and progenitor cells is altering the therapeutic landscape for patients with hematologic, immunologic, and metabolic disorders but has not yet been successfully developed for individuals with the bone marrow failure syndrome Diamond-Blackfan anemia (DBA). More than 30 mutations cause DBA through impaired ribosome function and lead to inefficient translation of the erythroid master regulator GATA1, providing a potential avenue for therapeutic intervention applicable to all patients with DBA, irrespective of the underlying genotype. Here, we report the development of a clinical-grade lentiviral gene therapy that achieves erythroid lineage-restricted expression of GATA1. We show that this vector is capable of augmenting erythropoiesis in DBA models and diverse patient samples without impacting hematopoietic stem cell function or demonstrating any signs of premalignant clonal expansion. These preclinical safety and efficacy data provide strong support for the first-in-human universal gene therapy trial for DBA through regulated GATA1 expression.

Keywords:  Diamond-Blackfan anemia; GATA1; bone marrow failure; enhancer; erythropoiesis; gene therapy; hematopoiesis; hematopoietic stem cell; hypoplastic anemia; lentivirus

DOI:  https://doi.org/10.1016/j.stem.2024.10.012
Cell Rep Med. 2024 Nov 08. pii: S2666-3791(24)00597-4. [Epub ahead of print] 101826

IL-21/IL-21R signaling renders acute myeloid leukemia stem cells more susceptible to cytarabine treatment and CAR T cell therapy.

Viviana Rubino, Michelle Hüppi, Sabine Höpner, Luigi Tortola, Noah Schnüriger, Hugo Legenne, Lea Taylor, Svenja Voggensperger, Irene Keller, Remy Bruggman, Marie-Noëlle Kronig, Ulrike Bacher, Manfred Kopf, Adrian F Ochsenbein, Carsten Riether.

  Self-renewal programs in leukemia stem cells (LSCs) predict poor prognosis in patients with acute myeloid leukemia (AML). We identify CD4+ T cell-derived interleukin (IL)-21 as an important negative regulator of self-renewal of LSCs. IL-21/IL-21R signaling favors asymmetric cell division and differentiation in LSCs through the activation of p38-MAPK signaling, resulting in reduced LSC numbers and significantly prolonged survival in murine AML models. In human AML, serum IL-21 at diagnosis is identified as an independent positive prognostic biomarker for outcome and correlates with improved survival and higher complete remission rates in patients that underwent high-dose chemotherapy. IL-21 treatment inhibits primary LSC function and enhances the effect of cytarabine and CD70 CAR T cell treatment on LSCs in vitro. Low-dose IL-21 treatment prolongs the survival of AML mice in syngeneic and xenograft experiments. Therefore, promoting IL-21/IL-21R signaling on LSCs may be an approach to reduce stemness and increase differentiation in AML.

Keywords:  AML; CD70-targeting CAR T cells; IL-21; cytarabine; leukemia stem cell; p38 MAPK; self-renewal; stemness

DOI:  https://doi.org/10.1016/j.xcrm.2024.101826
J Clin Invest. 2024 Sep 17. pii: e180738. [Epub ahead of print]134(22):

CXCL8 secreted by immature granulocytes inhibits WT hematopoiesis in chronic myelomonocytic leukemia.

Paul Deschamps, Margaux Wacheux, Axel Gosseye, Margot Morabito, Arnaud Pagès, Anne-Marie Lyne, Alexia Alfaro, Philippe Rameau, Aygun Imanci, Rabie Chelbi, Valentine Marchand, Aline Renneville, Mrinal M Patnaik, Valerie Lapierre, Bouchra Badaoui, Orianne Wagner-Ballon, Céline Berthon, Thorsten Braun, Christophe Willekens, Raphael Itzykson, Pierre Fenaux, Sylvain Thépot, Gabriel Etienne, Emilie Elvira-Matelot, Francoise Porteu, Nathalie Droin, Leïla Perié, Lucie Laplane, Eric Solary, Dorothée Selimoglu-Buet.

  Chronic myelomonocytic leukemia (CMML) is a severe myeloid malignancy with limited therapeutic options. Single-cell analysis of clonal architecture demonstrates early clonal dominance with few residual WT hematopoietic stem cells. Circulating myeloid cells of the leukemic clone and the cytokines they produce generate a deleterious inflammatory climate. Our hypothesis is that therapeutic control of the inflammatory component in CMML could contribute to stepping down disease progression. The present study explored the contribution of immature granulocytes (iGRANs) to CMML progression. iGRANs were detected and quantified in the peripheral blood of patients by spectral and conventional flow cytometry. Their accumulation was a potent and independent poor prognostic factor. These cells belong to the leukemic clone and behaved as myeloid-derived suppressor cells. Bulk and single-cell RNA-Seq revealed a proinflammatory status of iGRAN that secreted multiple cytokines of which CXCL8 was at the highest level. This cytokine inhibited the proliferation of WT but not CMML hematopoietic stem and progenitor cells (HSPCs) in which CXCL8 receptors were downregulated. CXCL8 receptor inhibitors and CXCL8 blockade restored WT HSPC proliferation, suggesting that relieving CXCL8 selective pressure on WT HSPCs is a potential strategy to slow CMML progression and restore some healthy hematopoiesis.

Keywords:  Cytokines; Hematology; Leukemias; Neutrophils

DOI:  https://doi.org/10.1172/JCI180738
Nature. 2024 Nov 13.

Adult skull bone marrow is an expanding and resilient haematopoietic reservoir.

Bong Ihn Koh, Vishal Mohanakrishnan, Hyun-Woo Jeong, Hongryeol Park, Kai Kruse, Young Jun Choi, Melina Nieminen-Kelhä, Rahul Kumar, Raquel S Pereira, Susanne Adams, Hyuek Jong Lee, M Gabriele Bixel, Peter Vajkoczy, Daniela S Krause, Ralf H Adams.

The bone marrow microenvironment is a critical regulator of haematopoietic stem cell self-renewal and fate1. Although it is appreciated that ageing, chronic inflammation and other insults compromise bone marrow function and thereby negatively affect haematopoiesis2, it is not known whether different bone compartments exhibit distinct microenvironmental properties and functional resilience. Here we use imaging, pharmacological approaches and mouse genetics to uncover specialized properties of bone marrow in adult and ageing skull. Specifically, we show that the skull bone marrow undergoes lifelong expansion involving vascular growth, which results in an increasing contribution to total haematopoietic output. Furthermore, skull is largely protected against major hallmarks of ageing, including upregulation of pro-inflammatory cytokines, adipogenesis and loss of vascular integrity. Conspicuous rapid and dynamic changes to the skull vasculature and bone marrow are induced by physiological alterations, namely pregnancy, but also pathological challenges, such as stroke and experimental chronic myeloid leukaemia. These responses are highly distinct from femur, the most extensively studied bone marrow compartment. We propose that skull harbours a protected and dynamically expanding bone marrow microenvironment, which is relevant for experimental studies and, potentially, for clinical treatments in humans.

DOI: https://doi.org/10.1038/s41586-024-08163-9
Life Sci Alliance. 2025 Jan;pii: e202402760. [Epub ahead of print]8(1):

Mass spectrometry-based proteomic exploration of diverse murine macrophage cellular models.

Jack Gudgeon, Abeer Dannoura, Ritika Chatterjee, Frances Sidgwick, Benjamin Ba Raymond, Andrew M Frey, José Luis Marin-Rubio, Matthias Trost.

Immortalised cell lines that mimic their primary cell counterparts are fundamental to research, particularly when large cell numbers are required. Here, we report that immortalisation of bone marrow-derived macrophages (iBMDMs) using the J2 virus resulted in the loss of a protein of interest, MSR1, in WT cells by an unknown mechanism. This led us to perform an in-depth mass spectrometry-based proteomic characterisation of common murine macrophage cell lines (J774A.1, RAW264.7, and BMA3.1A7), in comparison with the iBMDMs, as well as primary BMDMs from both C57BL/6 and BALB/c mice. This analysis revealed striking differences in protein profiles associated with macrophage polarisation, phagocytosis, pathogen recognition, and interferon signalling. Among the cell lines, J774A.1 cells were the most similar to the gold standard primary BMDM model, whereas BMA3.1A7 cells were the least similar because of the reduction in abundance of several key proteins related closely to macrophage function. This comprehensive proteomic dataset offers valuable insights into the use and suitability of macrophage cell lines for cell signalling and inflammation research.

DOI: https://doi.org/10.26508/lsa.202402760
Nat Commun. 2024 Nov 13. 15(1): 9820

Succinate dehydrogenase deficiency-driven succinate accumulation induces drug resistance in acute myeloid leukemia via ubiquitin-cullin regulation.

Yifan Chen, Miao Xian, Wenwen Ying, Jiayi Liu, Shaowei Bing, Xiaomin Wang, Jiayi Yu, Xiaojun Xu, Senfeng Xiang, Xuejing Shao, Ji Cao, Qiaojun He, Bo Yang, Meidan Ying.

Drug resistance is vital for the poor prognosis of acute myeloid leukemia (AML) patients, but the underlying mechanism remains poorly understood. Given the unique microenvironment of bone marrow, we reasoned that drug resistance of AML might rely on distinct metabolic processes. Here, we identify succinate dehydrogenase (SDH) deficiency and over-cumulative succinate as typical features in AML, with a marked function in causing the resistance of AML cells to various anti-cancer therapies. Mechanistically, succinate promotes the accumulation of oncogenic proteins in a manner that precedes transcriptional activation. This function is mediated by succinate-triggered upregulation of ubiquitin-conjugating enzyme E2M (UBC12) phosphorylation, which impairs its E2 function in cullins neddylation. Notably, decreasing succinate by fludarabine can restore the sensitivity of anti-cancer drugs in SDH-deficient AML. Together, we uncover the function of succinate in driving drug resistance by regulating p-UBC12/cullin activity, and indicate reshaping succinate metabolism as a promising treatment for SDH-deficient AML.

DOI: https://doi.org/10.1038/s41467-024-53398-9
Nat Commun. 2024 Nov 08. 15(1): 9697

PU.1 eviction at lymphocyte-specific chromatin domains mediates glucocorticoid response in acute lymphoblastic leukemia.

Dominik Beck, Honghui Cao, Feng Tian, Yizhou Huang, Miao Jiang, Han Zhao, Xiaolu Tai, Wenqian Xu, Hansen J Kosasih, David J Kealy, Weiye Zhao, Samuel J Taylor, Timothy A Couttas, Gaoxian Song, Diego Chacon-Fajardo, Yashna Walia, Meng Wang, Adam A Dowle, Andrew N Holding, Katherine S Bridge, Chao Zhang, Jin Wang, Jian-Qing Mi, Richard B Lock, Charles E de Bock, Duohui Jing.

The epigenetic landscape plays a critical role in cancer progression, yet its therapeutic potential remains underexplored. Glucocorticoids are essential components of treatments for lymphoid cancers, but resistance, driven in part by epigenetic changes at glucocorticoid-response elements, poses a major challenge to effective therapies. Here we show that glucocorticoid treatment induces distinct patterns of chromosomal organization in glucocorticoid-sensitive and resistant acute lymphoblastic leukemia xenograft models. These glucocorticoid-response elements are primed by the pioneer transcription factor PU.1, which interacts with the glucocorticoid receptor. Eviction of PU.1 promotes receptor binding, increasing the expression of genes involved in apoptosis and facilitating a stronger therapeutic response. Treatment with a PU.1 inhibitor enhances glucocorticoid sensitivity, demonstrating the clinical potential of targeting this pathway. This study uncovers a mechanism involving PU.1 and the glucocorticoid receptor, linking transcription factor activity with drug response, and suggesting potential therapeutic strategies for overcoming resistance.

DOI: https://doi.org/10.1038/s41467-024-54096-2
Biochim Biophys Acta Mol Basis Dis. 2024 Nov 08. pii: S0925-4439(24)00559-3. [Epub ahead of print] 167565

GPR68 supports AML cells through the calcium/calcineurin pro-survival pathway and confers chemoresistance by mediating glucose metabolic symbiosis.

Xiaofei He, Caleb Hawkins, Lauren Lawley, Tra Mi Phan, Isaac Park, Nicole Joven, Jiajia Zhang, Mark Wunderlich, Benjamin Mizukawa, Shanshan Pei, Amisha Patel, Jennifer VanOudenhove, Stephanie Halene, Jing Fang.

  Accumulating evidence demonstrates that the "Warburg effect" that glycolysis is enhanced even in the presence of oxygen existed in hematopoietic malignancies, contributing to extracellular acidosis. G-protein coupled receptor 68 (GPR68), as a proton sensing GPCR responding to extracellular acidosis, is expected to play a critical role in hematopoietic malignancies. In the present study, we found that GPR68 was overexpressed in acute myeloid leukemia (AML) cells, and GPR68 deficiency impaired AML cell survival in vitro and cell engraftment in vivo. Mechanistic studies revealed that unlike GPR68 regulates Calpain1 in myelodysplastic syndromes (MDS) cells, GPR68 deficiency reduced cytosolic Ca2+ levels and calcineurin (CaN) activity in AML cells through an NFAT-independent mechanism. Moreover, the decreased Ca2+ levels disturbed cellular respiration (i.e., oxidative phosphorylation, OxPhos) by inhibiting isocitrate dehydrogenase (IDH) activity; this was more pronounced when BCL2 was inhibited simultaneously. Interestingly, GPR68 inhibition also decreased aerobic glycolysis in AML cells in a Ca2+-independent manner, suggesting that GPR68 mediated glucose metabolic symbiosis. As glucose metabolic symbiosis and the heterogeneous dependencies on aerobic glycolysis and cellular respiration tremendously impact chemosensitivity, the inhibition of GPR68 potentiated the tumoricidal effect of first-line chemotherapeutic agents, including BCL-2 inhibitors targeting OxPhos and cytarabine (AraC) targeting glycolysis. Consistent with these in vitro observations, higher levels of GPR68 were associated with inferior clinical outcomes in AML patients who received chemotherapies. In short, GPR68 drives the Ca2+/CaN pro-survival pathway and mediates glucose metabolic pathways in AML cells. Targeting GPR68 eradicates AML cells and alleviates chemoresistance, which could be exploited as a therapeutic target. The overexpression of GPR68 drives a Ca2+/CaN pro-survival pathway and mediates glucose metabolic symbiosis in AML cells, suggesting the diagnostic and therapeutic potential of GPR68 in AML. (GPR68, G proton-coupled receptor 68; PLCβ, phospholipase C beta; CaN, Calcineurin; IDH, isocitrate dehydrogenase; HIF-1α, Hypoxia-inducible factor alpha subunit; GLUT1, Glucose transporter type 1; HK-1, Hexokinase 1).

Keywords:  Calcineurin; Calcium; Chemoresistance; Glucose metabolic symbiosis; Proton-sensing GPR68

DOI:  https://doi.org/10.1016/j.bbadis.2024.167565
Cell Rep Med. 2024 Nov 08. pii: S2666-3791(24)00594-9. [Epub ahead of print] 101823

A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells.

Lucrezia Della Volpe, Federico Midena, Roberta Vacca, Teresa Tavella, Laura Alessandrini, Giacomo Farina, Chiara Brandas, Elena Lo Furno, Kety Giannetti, Edoardo Carsana, Matteo M Naldini, Matteo Barcella, Samuele Ferrari, Stefano Beretta, Antonella Santoro, Simona Porcellini, Angelica Varesi, Diego Gilioli, Anastasia Conti, Ivan Merelli, Bernhard Gentner, Anna Villa, Luigi Naldini, Raffaella Di Micco.

  Ex vivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here, we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks, enhancing the reconstitution capacity of edited HSPCs. However, this results in lower HDR efficiency, rendering ex vivo culture necessary yet detrimental. Mechanistically, ex vivo activation triggers a multi-step process initiated by p38 mitogen-activated protein kinase (MAPK) phosphorylation, which generates mitogenic reactive oxygen species (ROS), promoting fast cell-cycle progression and subsequent proliferation-induced DNA damage. Thus, p38 inhibition before gene editing delays G1/S transition and expands transcriptionally defined HSCs, ultimately endowing edited cells with superior multi-lineage differentiation, persistence throughout serial transplantation, enhanced polyclonal repertoire, and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications.

Keywords:  CRISPR-Cas9; DNA damage; DNA damage response; cell cycle; clonal output; differentiation; gene editing; hematopoietic stem cells; p38 MAPK-ROS; proliferative stress; single-cell analyses

DOI:  https://doi.org/10.1016/j.xcrm.2024.101823
Stem Cell Reports. 2024 Sep 25. pii: S2213-6711(24)00266-2. [Epub ahead of print]

Interleukin-1β induces trained innate immunity in human hematopoietic progenitor cells in vitro.

Daniela Flores-Gomez, Willemijn Hobo, Diede van Ens, Elise L Kessler, Boris Novakovic, Nicolaas P M Schaap, Wim H C Rijnen, Leo A B Joosten, Mihai G Netea, Niels P Riksen, Siroon Bekkering.

  Innate immune cells can develop a long-lasting hyperresponsive phenotype, termed trained immunity, mediated by epigenetic and metabolic reprogramming. In mice, exposure to Bacille Calmette-Guérin (BCG), β-glucan, or Western diet induces trained immunity by reprogramming hematopoietic progenitor cells (HPCs), through interleukin-1β (IL-1β) signaling in the bone marrow (BM). We investigated whether IL-1β induces trained immunity in primary human BM-derived HPCs in vitro. We exposed human BM-derived HPCs to IL-1β for 4 h. HPCs were expanded and differentiated into monocytes followed by functional and transcriptomic characterization. IL-1β-exposed HPCs showed higher granulocyte-macrophage colony-forming units. The monocyte offspring produced more tumor necrosis factor (TNF) and IL-1β after restimulation with lipopolysaccharide (LPS) and Pam3Cys and is metabolically more active. Transcriptomic analysis showed upregulation of key atherogenic and inflammatory pathways. In conclusion, brief exposure of human BM-derived HPCs to IL-1β in vitro induces a trained immunity phenotype.

Keywords:  IL-1β; bone marrow; hematopoietic progenitor cells; macrophages; monocytes; trained immunity

DOI:  https://doi.org/10.1016/j.stemcr.2024.09.004
EMBO J. 2024 Nov 14.

Redistribution of PU.1 partner transcription factor RUNX1 binding secures cell survival during leukemogenesis.

Alexander Bender, Füsun Boydere, Ashok Kumar Jayavelu, Alessia Tibello, Thorsten König, Hanna Aleth, Gerd Meyer Zu Hörste, Thomas Vogl, Frank Rosenbauer.

  Transcription factors (TFs) orchestrating lineage-development often control genes required for cellular survival. However, it is not well understood how cells survive when such TFs are lost, for example in cancer. PU.1 is an essential TF for myeloid fate, and mice with downregulated PU.1 levels develop acute myeloid leukemia (AML). Combining a multi-omics approach with a functional genetic screen, we reveal that PU.1-downregulated cells fundamentally change their survival control from cytokine-driven pathways to overexpression of an autophagy-predominated stem cell gene program, for which we also find evidence in human AML. Control of this program involves redirected chromatin occupancy of the PU.1 partner TF Runx1 to a lineage-inappropriate binding site repertoire. Hence, genomic reallocation of TF binding upon loss of a partner TF can act as a pro-oncogenic failsafe mechanism by sustaining cell survival during leukemogenesis.

Keywords:  Myeloid Development; Myeloid Leukemia; PU.1; RUNX1

DOI:  https://doi.org/10.1038/s44318-024-00295-y
Clin Transl Med. 2024 Nov;14(11): e70085

Molecular characterization of human HSPCs with different cell fates in vivo using single-cell transcriptome analysis and lentiviral barcoding technology.

Junnan Hua, Ke Wang, Yue Chen, Xiaojing Xu, Guoyi Dong, Yue Li, Rui Liu, Yecheng Xiong, Jiabin Ding, Tingting Zhang, Xinru Zeng, Yuxi Li, Haixi Sun, Ying Gu, Sixi Liu, Wenjie Ouyang, Chao Liu.

  Hematopoietic stem and progenitor cells (HSPCs) possess the potential to produce all types of blood cells throughout their lives. It is well recognized that HSPCs are heterogeneous, which is of great significance for their clinical applications and the treatment of diseases associated with HSPCs. This study presents a novel technology called Single-Cell transcriptome Analysis and Lentiviral Barcoding (SCALeBa) to investigate the molecular mechanisms underlying the heterogeneity of human HSPCs in vivo. The SCALeBa incorporates a transcribed barcoding library and algorithm to analyze the individual cell fates and their gene expression profiles simultaneously. Our findings using SCALeBa reveal that HSPCs subset with stronger stemness highly expressed MYL6B, ATP2A2, MYO19, MDN1, ING3, and so on. The high expression of COA3, RIF1, RAB14, and GOLGA4 may contribute to the pluripotent-lineage differentiation of HSPCs. Moreover, the roles of the representative genes revealed in this study regarding the stemness of HPSCs were confirmed with biological experiments. HSPCs expressing MRPL23 and RBM4 genes may contribute to differentiation bias into myeloid and lymphoid lineage, respectively. In addition, transcription factor (TF) characteristics of lymphoid and myeloid differentiation bias HSPCs subsets were identified and linked to previously identified genes. Furthermore, the stemness, pluripotency, and differentiation-bias genes identified with SCALeBa were verified in another independent HSPCs dataset. Finally, this study proposes using the SCALeBa-generated tracking trajectory to improve the accuracy of pseudo-time analysis results. In summary, our study provides valuable insights for understanding the heterogeneity of human HSPCs in vivo and introduces a novel technology, SCALeBa, which holds promise for broader applications. KEY POINTS: SCALeBa and its algorithm are developed to study the molecular mechanism underlying human HSPCs identity and function. The human HSPCs expressing MYL6B, MYO19, ATP2A2, MDN1, ING3, and PHF20 may have the capability for high stemness. The human HSPCs expressing COA3, RIF1, RAB14, and GOLGA4 may have the capability for pluripotent-lineage differentiation. The human HSPCs expressing MRPL23 and RBM4 genes may have the capability to differentiate into myeloid and lymphoid lineage respectively in vivo. The legitimacy of the identified genes with SCALeBa was validated using biological experiments and a public human HSPCs dataset. SCALeBa improves the accuracy of differentiation trajectories in monocle2-based pseudo-time analysis.

Keywords:  barcoding technology; hematopoietic stem and progenitor cells; lineage tracing; scRNA‐seq

DOI:  https://doi.org/10.1002/ctm2.70085
J Cell Biol. 2025 Feb 03. pii: e202403136. [Epub ahead of print]224(2):

Structural response of microtubule and actin cytoskeletons to direct intracellular load.

Ryota Orii, Hirokazu Tanimoto.

Microtubule and actin are the two major cytoskeletal polymers that form organized functional structures in the interior of eukaryotic cells. Although the structural mechanics of the cytoskeleton has been extensively studied by direct manipulations in in vitro reconstitution systems, such unambiguous characterizations inside the living cell are sparse. Here, we report a comprehensive analysis of how the microtubule and actin cytoskeletons structurally respond to direct intracellular load. Ferrofluid-based intracellular magnetic tweezers reveal rheological properties of the microtubule complex primarily determined by filamentous actin. The strain fields of the microtubule complex and actin meshwork follow the same scaling, suggesting that the two cytoskeletal systems behave as an integrated elastic body. The structural responses of single microtubules to contact and remote forces further evidence that the individual microtubules are enclosed by the elastic medium of actin. These results, directly characterizing the microtubule and actin cytoskeletons as an interacting continuum throughout the cytoplasm, serve as a cornerstone for the physical understanding of intracellular organization.

DOI: https://doi.org/10.1083/jcb.202403136
Proc Natl Acad Sci U S A. 2024 Nov 19. 121(47): e2413837121

Parallel phosphoproteomics and metabolomics map the global metabolic tyrosine phosphoproteome.

Alissandra L Hillis, Tigist Tamir, Grace E Perry, John M Asara, Jared L Johnson, Tomer M Yaron, Lewis C Cantley, Forest M White, Alex Toker.

  Tyrosine phosphorylation of metabolic enzymes is an evolutionarily conserved posttranslational modification that facilitates rapid and reversible modulation of enzyme activity, localization, or function. Despite the high abundance of tyrosine phosphorylation events detected on metabolic enzymes in high-throughput mass spectrometry-based studies, functional characterization of tyrosine phosphorylation sites has been limited to a subset of enzymes. Since tyrosine phosphorylation is dysregulated across human diseases, including cancer, understanding the consequences of metabolic enzyme tyrosine phosphorylation events is critical for informing disease biology and therapeutic interventions. To globally identify metabolic enzyme tyrosine phosphorylation events and simultaneously assign functional significance to these sites, we performed parallel phosphoproteomics and polar metabolomics in nontumorigenic mammary epithelial cells (MCF10A) stimulated with epidermal growth factor (EGF) in the absence or presence of the EGF receptor inhibitor erlotinib. We performed an integrated analysis of the phosphoproteomic and metabolomic datasets to identify tyrosine phosphorylation sites on metabolic enzymes with functional consequences. We identified two previously characterized (pyruvate kinase muscle isozyme, phosphoglycerate mutase 1) and two uncharacterized (glutathione S-transferase Pi 1, glutamate dehydrogenase 1) tyrosine phosphorylation sites on metabolic enzymes with purported functions based on metabolomic analyses. We validated these hits using a doxycycline-inducible CRISPR interference system in MCF10A cells, in which target metabolic enzymes were depleted with simultaneous reexpression of wild-type, phosphomutant, or phosphomimetic isoforms. Together, these data provide a framework for identification, prioritization, and characterization of tyrosine phosphorylation sites on metabolic enzymes with functional significance.

Keywords:  EGFR; cancer metabolism; metabolomics; phosphotyrosine; proteomics

DOI:  https://doi.org/10.1073/pnas.2413837121
J Hematol Oncol. 2024 Nov 07. 17(1): 107

A single-cell transcriptomic map of the murine and human multiple myeloma immune microenvironment across disease stages.

Emma Verheye, Daliya Kancheva, Hatice Satilmis, Niels Vandewalle, Rong Fan, Pauline M R Bardet, Emile J Clappaert, Kevin Verstaen, Ann De Becker, Karin Vanderkerken, Kim De Veirman, Damya Laoui.

  BACKGROUND: The long-term effectiveness of immunotherapies against Multiple Myeloma (MM) remains elusive, demonstrated by the inevitable relapse in patients. This underscores the urgent need for an in-depth analysis of the MM tumor-immune microenvironment (TME). Hereto, a representative immunocompetent MM mouse model can offer a valuable approach to study the dynamic changes within the MM-TME and to uncover potential resistance mechanisms hampering effective and durable therapeutic strategies in MM.METHODS: We generated a comprehensive single-cell RNA-sequencing atlas of the MM-TME in bone marrow and spleen encompassing different stages of disease, using the immunocompetent 5T33MM mouse model. Through comparative analysis, we correlated our murine dataset with the pathogenesis in MM patients by reanalyzing publicly available datasets of human bone marrow samples across various disease stages. Using flow cytometry, we validated the dynamic changes upon disease progression in the 5T33MM model. Furthermore, interesting target populations, as well as the immune-boosting anti-CD40 agonist (αCD40) therapy were tested ex vivo on murine and human primary samples and in vivo using the 5T33MM model.
RESULTS: In this study, we identified the heterogenous and dynamic changes within the TME of murine and human MM. We found that the MM-TME was characterized by an increase in T cells, accompanied with an exhausted phenotype. Although neutrophils appeared to be rather innocuous at early disease stages, they acquired a pro-tumorigenic phenotype during MM progression. Moreover, conventional dendritic cells (cDCs) showed a less activated phenotype in MM, underscoring the potential of immune-boosting therapies such as αCD40 therapy. Importantly, we provided the first pre-clinical evaluation of αCD40 therapy and demonstrated successful induction of cDC- and T-cell activation, accompanied by a significant short-term anti-tumor response.
CONCLUSIONS: This resource provides a comprehensive and detailed immune atlas of the evolution in human and murine MM disease progression. Our findings can contribute to immune-based patient stratification and facilitate the development of novel and durable (immune) therapeutic strategies in MM.

Keywords:  5T33MM immune microenvironment; Anti-CD40 agonist therapy; Human-mouse comparison; Multiple myeloma progression; Single-cell RNA-sequencing

DOI:  https://doi.org/10.1186/s13045-024-01629-3
J Biol Chem. 2024 Nov 08. pii: S0021-9258(24)02479-7. [Epub ahead of print] 107977

Redox Regulation of Proteostasis.

Long Duy Duong, James D West, Kevin A Morano.

  Oxidants produced through endogenous metabolism or encountered in the environment react directly with reactive sites in biological macromolecules. Many proteins, in particular, are susceptible to oxidative damage, which can lead their altered structure and function. Such structural and functional changes trigger a cascade of events that influence key components of the proteostasis network. Here, we highlight recent advances in our understanding of how cells respond to the challenges of protein folding and metabolic alterations that occur during oxidative stress. Immediately after an oxidative insult, cells selectively block the translation of most new proteins and shift molecular chaperones from a folding to a holding role to prevent wholesale protein aggregation. At the same time, adaptive responses in gene expression are induced, allowing for increased expression of antioxidant enzymes, enzymes that carry out reduction of oxidized proteins, and molecular chaperones, all of which serve to mitigate oxidative damage and rebalance proteostasis. Likewise, concomitant activation of protein clearance mechanisms, namely proteasomal degradation and particular autophagic pathways, promotes degradation of irreparably damaged proteins. As oxidative stress is associated with inflammation, aging, and numerous age-related disorders, the molecular events described herein are therefore major determinants of health and disease.

Keywords:  chaperone; foldase; heat shock protein; holdase; oxidation; oxidative stress; post-translational modification; protein degradation; proteostasis; redox regulation; thiol modification; transcriptional response; translation repression

DOI:  https://doi.org/10.1016/j.jbc.2024.107977
Cancer Res. 2024 Nov 08.

Modeling Drug Responses and Evolutionary Dynamics using Patient-Derived Xenografts Reveals Precision Medicine Strategies for Triple Negative Breast Cancer.

Abigail Shea, Yaniv Eyal-Lubling, Daniel Guerrero-Romero, Raquel Manzano Garcia, Wendy Greenwood, Martin O'Reilly, Dimitra Georgopoulou, Maurizio Callari, Giulia Lerda, Sophia Wix, Agnese Giovannetti, Riccardo Masina, Elham Esmaeilishirazifard, Wei Cope, Alistair G Martin, Ai Nagano, Lisa Young, Steven Kupczak, Yi Cheng, Helen Bardwell, Elena Provenzano, Justine Kane, Jonny Lay, Louise Grybowicz, Karen McAdam, Carlos Caldas, Jean Abraham, Oscar M Rueda, Alejandra Bruna.

The inter- and intra-tumor heterogeneity of triple negative breast cancers (TNBC), which is reflected in diverse drug responses, interplays with tumor evolution. Here, we developed a preclinical experimental and analytical framework using treatment-naive TNBC patient-derived tumor xenografts (PDTX) to test their predictive value in personalized cancer treatment approaches. Patients and their matched PDTX exhibited concordant drug responses to neoadjuvant therapy using two trial designs and dosing schedules. This platform enabled analysis of non-genetic mechanisms involved in relapse dynamics. Treatment resulted in permanent phenotypic changes with functional and therapeutic consequences. High throughput drug screening methods in ex vivo patient derived tumor xenograft cells (PDTCs) revealed patient-specific drug response changes dependent on first-line therapy. This was validated in vivo, as exemplified by a change in olaparib sensitivity in tumors previously treated with clinically relevant cycles of standard-of-care chemotherapy. In summary, PDTXs provide a robust tool to test patient drug responses and therapeutic regimens and to model evolutionary trajectories. However, high inter-model variability and permanent non-genomic transcriptional changes constrain their use for personalized cancer therapy. This work highlights important considerations associated with preclinical drug response modeling and potential uses of the platform to identify efficacious and preferential sequential therapeutic regimens.

DOI: https://doi.org/10.1158/0008-5472.CAN-24-1703
Mol Cell. 2024 Nov 05. pii: S1097-2765(24)00862-1. [Epub ahead of print]

VPS4A is the selective receptor for lipophagy in mice and humans.

Debajyoti Das, Mridul Sharma, Deepanshi Gahlot, Shervin S Nia, Chandrima Gain, Matthew Mecklenburg, Z Hong Zhou, Mathieu Bourdenx, Lipi Thukral, Nuria Martinez-Lopez, Rajat Singh.

  Lipophagy is a ubiquitous mechanism for degradation of lipid droplets (LDs) in lysosomes. Autophagy receptors selectively target organelles for lysosomal degradation. The selective receptor for lipophagy remains elusive. Using mouse liver phosphoproteomics and human liver transcriptomics, we identify vacuolar-protein-sorting-associated protein 4A (VPS4A), a member of a large family AAA+ ATPases, as a selective receptor for lipophagy. We show that phosphorylation of VPS4A on Ser95,97 and its localization to LDs in response to fasting drives lipophagy. Imaging/three-dimensional (3D) reconstruction and biochemical analyses reveal the concomitant degradation of VPS4A and LDs in lysosomes in an autophagy-gene-7-sensitive manner. Either silencing VPS4A or targeting VPS4AS95,S97 phosphorylation or VPS4A binding to LDs or LC3 blocks lipophagy without affecting other forms of selective autophagy. Finally, VPS4A levels and markers of lipophagy are markedly reduced in human steatotic livers-revealing a fundamental role of VPS4A as the lipophagy receptor in mice and humans.

Keywords:  MASLD; VPS4A; autophagy; human; lipid droplet; lipophagy; liver; lysosome; phosphorylation; receptor

DOI:  https://doi.org/10.1016/j.molcel.2024.10.022
Nat Commun. 2024 Nov 12. 15(1): 9797

HSF-1 promotes longevity through ubiquilin-1-dependent mitochondrial network remodelling.

Annmary Paul Erinjeri, Xunyan Wang, Rhianna Williams, Riccardo Zenezini Chiozzi, Konstantinos Thalassinos, Johnathan Labbadia.

Increased activity of the heat shock factor, HSF-1, suppresses proteotoxicity and enhances longevity. However, the precise mechanisms by which HSF-1 promotes lifespan are unclear. Using an RNAi screen, we identify ubiquilin-1 (ubql-1) as an essential mediator of lifespan extension in worms overexpressing hsf-1. We find that hsf-1 overexpression leads to transcriptional downregulation of all components of the CDC-48-UFD-1-NPL-4 complex, which is central to both endoplasmic reticulum and mitochondria associated protein degradation, and that this is complemented by UBQL-1-dependent turnover of NPL-4.1. As a consequence, mitochondrial network dynamics are altered, leading to increased lifespan. Together, our data establish that HSF-1 mediates lifespan extension through mitochondrial network adaptations that occur in response to down-tuning of components associated with organellar protein degradation pathways.

DOI: https://doi.org/10.1038/s41467-024-54136-x
Hemasphere. 2024 Nov;8(11): e70039

Preclinical evaluation of the CD38-targeting engineered toxin body MT-0169 against multiple myeloma.

Wassilis S C Bruins, Rosa Rentenaar, John Newcomb, Wenrou Zheng, Ruud W J Ruiter, Thomas Baardemans, Eric Poma, Chris Moore, Garrett L Robinson, Anya Lublinsky, Yuhong Zhang, Sakeena Syed, Michael Milhollen, Ajeeta B Dash, Niels W C J van de Donk, Richard W J Groen, Sonja Zweegman, Tuna Mutis.

Despite significant progress in the treatment of multiple myeloma (MM), relapsed/refractory patients urgently require more effective therapies. We here describe the discovery, mechanism of action, and preclinical anti-MM activity of engineered toxin body MT-0169, a next-generation immunotoxin comprising a CD38-specific antibody fragment linked to a de-immunized Shiga-like toxin A subunit (SLTA) payload. We show that specific binding of MT-0169 to CD38 on MM cell lines triggers rapid internalization of SLTA, causing cell death via irreversible ribosome inhibition, protein synthesis blockade, and caspase 3/7 activation. In co-culture experiments, bone marrow mesenchymal stromal cells did not induce drug resistance against MT-0169. In the preclinical setting, MT-0169 effectively lysed primary MM cells from newly diagnosed and heavily pretreated MM patients, including those refractory to daratumumab, with minimal toxicity against nonmalignant hematopoietic cells. MM cell lysis showed a significant correlation with their CD38 expression levels but not with cytogenetic risk, tumor load, or number of prior lines of therapy. Finally, MT-0169 showed efficient in vivo anti-MM activity in various mouse xenograft models, including one in which MM cells are grown in a humanized bone marrow-like niche. These findings support clinical investigation of MT-0169 in relapsed/refractory MM patients, including those refractory to CD38-targeting immunotherapies.

DOI: https://doi.org/10.1002/hem3.70039
Cytometry B Clin Cytom. 2024 Nov 13.

Improved identification of clinically relevant Acute Leukemia subtypes using standardized EuroFlow panels versus non-standardized approach.

Rafik Terra, Vincent Éthier, Lambert Busque, Ariane Morin-Quintal, Giovanni D'Angelo, Josée Hébert, Xuehai Wang, Guylaine Lépine, Richard LeBlanc, Julie Bergeron.

  Rare acute leukemia (AL) components or subtypes such as blastic plasmacytoid dendritic cell neoplasm (BPDCN) or early T-cell precursor acute Lymphoblastic Leukemia (ETP-ALL) can be difficult to detect by routine flow cytometry due to their immunophenotypes overlapping with other poorly differentiated AL. We hypothesized that using standardized EuroFlow™ Consortium approach could better diagnose such entities among cases that previously classified as acute myeloid leukemia (AML)-M0, AML with minimal differentiation, AML with myelodysplasia-related changes without further lineage differentiation, and AL of ambiguous lineage. In order to confirm this hypothesis and assess whether these AL subtypes such as BPDCN and ETP-ALL had previously gone undetected, we reanalyzed 49 banked cryopreserved sample cases using standardized EuroFlow™ Consortium panels. We also performed target sequencing to capture the mutational commonalities between these AL subtypes. Reanalysis led to revised or refined diagnoses for 23 cases (47%). Of these, five diagnoses were modified, uncovering 3 ETP-ALL and 2 typical BPDCN cases. In 12 AML cases, a variable proportion of immature plasmacytoid dendritic cell and/or monocytic component was newly identified. In one AML case, we have identified a megakaryoblastic differentiation. Finally, in five acute lymphoblastic leukemia (ALL) cases, we were able to more precisely determine the maturation stage. The application of standardized EuroFlow flow cytometry immunophenotyping improves the diagnostic accuracy of ALs and could impact treatment decisions.

Keywords:  EuroFlow standardized flow cytometry; acute leukemia; diagnosis; immunophenotype; plasmacytoid dendritic cell

DOI:  https://doi.org/10.1002/cyto.b.22213
Nat Commun. 2024 Nov 14. 15(1): 9865

Force-transducing molecular ensembles at growing microtubule tips control mitotic spindle size.

Lee-Ya Chu, Daniel Stedman, Julian Gannon, Susan Cox, Georgii Pobegalov, Maxim I Molodtsov.

Correct mitotic spindle size is required for accurate chromosome segregation during cell division. It is controlled by mechanical forces generated by molecular motors and non-motor proteins acting on spindle microtubules. However, how forces generated by individual proteins enable bipolar spindle organization is not well understood. Here, we develop tools to measure contributions of individual molecules to this force balance. We show that microtubule plus-end binding proteins act at microtubule tips synergistically with minus-end directed motors to produce a system that can generate both pushing and pulling forces. To generate pushing force, the system harnesses forces generated by the growing tips of microtubules providing unique contribution to the force balance distinct from all other motors that act in the mitotic spindle. Our results reveal that microtubules are essential force generators for establishing spindle size and pave the way for understanding how mechanical forces can be fine-tuned to control the fidelity of chromosome segregation.

DOI: https://doi.org/10.1038/s41467-024-54123-2
Nat Commun. 2024 Nov 09. 15(1): 9720

α-tubulin detyrosination fine-tunes kinetochore-microtubule attachments.

Hugo Girão, Joana Macário-Monteiro, Ana C Figueiredo, Ricardo Silva E Sousa, Elena Doria, Vladimir Demidov, Hugo Osório, Ariana Jacome, Patrick Meraldi, Ekaterina L Grishchuk, Helder Maiato.

Post-translational cycles of α-tubulin detyrosination and tyrosination generate microtubule diversity, the cellular functions of which remain largely unknown. Here we show that α-tubulin detyrosination regulates kinetochore-microtubule attachments to ensure normal chromosome oscillations and timely anaphase onset during mitosis. Remarkably, detyrosinated α-tubulin levels near kinetochore microtubule plus-ends depend on the direction of chromosome motion during metaphase. Proteomic analyses unveil that the KNL-1/MIS12/NDC80 (KMN) network that forms the core microtubule-binding site at kinetochores and the microtubule-rescue protein CLASP2 are enriched on tyrosinated and detyrosinated microtubules during mitosis, respectively. α-tubulin detyrosination enhances CLASP2 binding and NDC80 complex diffusion along the microtubule lattice in vitro. Rescue experiments overexpressing NDC80, including variants with slower microtubule diffusion, suggest a functional interplay with α-tubulin detyrosination for the establishment of a labile kinetochore-microtubule interface. These results offer a mechanistic explanation for how different detyrosinated α-tubulin levels near kinetochore microtubule plus-ends fine-tune load-bearing attachments to both growing and shrinking microtubules.

DOI: https://doi.org/10.1038/s41467-024-54155-8
J Cell Biol. 2024 Dec 02. pii: e202401084. [Epub ahead of print]223(12):

Non-cell autonomous regulation of cell-cell signaling and differentiation by mitochondrial ROS.

Yipeng Du, Lei Wang, Lizbeth Perez-Castro, Maralice Conacci-Sorrell, Matthew Sieber.

Mitochondrial reactive oxygen species (ROS) function intrinsically within cells to induce cell damage, regulate transcription, and cause genome instability. However, we know little about how mitochondrial ROS production non-cell autonomously impacts cell-cell signaling. Here, we show that mitochondrial dysfunction inhibits the plasma membrane localization of cell surface receptors that drive cell-cell communication during oogenesis. Within minutes, we found that mitochondrial ROS impairs exocyst membrane binding and leads to defective endosomal recycling. This endosomal defect impairs the trafficking of receptors, such as the Notch ligand Delta, during oogenesis. Remarkably, we found that overexpressing RAB11 restores ligand trafficking and rescues the developmental defects caused by ROS production. ROS production from adjacent cells acutely initiates a transcriptional response associated with growth and migration by suppressing Notch signaling and inducing extra cellualr matrix (ECM) remodeling. Our work reveals a conserved rapid response to ROS production that links mitochondrial dysfunction to the non-cell autonomous regulation of cell-cell signaling.

DOI: https://doi.org/10.1083/jcb.202401084
Nat Commun. 2024 Nov 08. 15(1): 9687

HURP regulates Kif18A recruitment and activity to synergistically control microtubule dynamics.

Juan M Perez-Bertoldi, Yuanchang Zhao, Akanksha Thawani, Ahmet Yildiz, Eva Nogales.

During mitosis, microtubule dynamics are regulated to ensure proper alignment and segregation of chromosomes. The dynamics of kinetochore-attached microtubules are regulated by hepatoma-upregulated protein (HURP) and the mitotic kinesin-8 Kif18A, but the underlying mechanism remains elusive. Using single-molecule imaging in vitro, we demonstrate that Kif18A motility is regulated by HURP. While sparse decoration of HURP activates the motor, higher concentrations hinder processive motility. To shed light on this behavior, we determine the binding mode of HURP to microtubules using cryo-EM. The structure helps rationalize why HURP functions as a microtubule stabilizer. Additionally, HURP partially overlaps with the microtubule-binding site of the Kif18A motor domain, indicating that excess HURP inhibits Kif18A motility by steric exclusion. We also observe that HURP and Kif18A function together to suppress dynamics of the microtubule plus-end, providing a mechanistic basis for how they collectively serve in microtubule length control.

DOI: https://doi.org/10.1038/s41467-024-53691-7
Nature. 2024 Nov 14.

Engineered receptors for soluble cellular communication and disease sensing.

Dan I Piraner, Mohamad H Abedi, Maria J Duran Gonzalez, Adam Chazin-Gray, Annie Lin, Iowis Zhu, Pavithran T Ravindran, Thomas Schlichthaerle, Buwei Huang, Tyler H Bearchild, David Lee, Sarah Wyman, Young-Wook Jun, David Baker, Kole T Roybal.

Despite recent advances in mammalian synthetic biology, there remains a lack of modular synthetic receptors that can robustly respond to soluble ligands and in turn activate bespoke cellular functions. Such receptors would have extensive clinical potential to regulate the activity of engineered therapeutic cells, but to date only receptors against cell surface targets have approached clinical translation1. To address this gap, we developed a receptor architecture called synthetic intramembrane proteolysis receptor (SNIPR), that has the added ability to be activated by soluble ligands, both natural and synthetic, with remarkably low baseline activity and high fold activation, through an endocytic, pH-dependent cleavage mechanism. We demonstrate the therapeutic capabilities of the receptor platform by localizing the activity of CAR T cells to solid tumors where soluble disease-associated factors are expressed, bypassing the major hurdle of on-target off-tumor toxicity in bystander organs. We further applied the SNIPR platform to engineer fully synthetic signaling networks between cells orthogonal to natural signaling pathways, expanding the scope of synthetic biology. Our design framework enables cellular communication and environmental interactions, extending the capabilities of synthetic cellular networking in clinical and research contexts.

DOI: https://doi.org/10.1038/s41586-024-08366-0
Science. 2024 Nov 15. 386(6723): eadh9215

A glutamine metabolic switch supports erythropoiesis.

Junhua Lyu, Zhimin Gu, Yuannyu Zhang, Hieu S Vu, Christophe Lechauve, Feng Cai, Hui Cao, Julia Keith, Valentina Brancaleoni, Francesca Granata, Irene Motta, Maria Domenica Cappellini, Lily Jun-Shen Huang, Ralph J DeBerardinis, Mitchell J Weiss, Min Ni, Jian Xu.

Metabolic requirements vary during development, and our understanding of how metabolic activity influences cell specialization is incomplete. Here, we describe a switch from glutamine catabolism to synthesis required for erythroid cell maturation. Glutamine synthetase (GS), one of the oldest functioning genes in evolution, is activated during erythroid maturation to detoxify ammonium generated from heme biosynthesis, which is up-regulated to support hemoglobin production. Loss of GS in mouse erythroid precursors caused ammonium accumulation and oxidative stress, impairing erythroid maturation and recovery from anemia. In β-thalassemia, GS activity is inhibited by protein oxidation, leading to glutamate and ammonium accumulation, whereas enhancing GS activity alleviates the metabolic and pathological defects. Our findings identify an evolutionarily conserved metabolic adaptation that could potentially be leveraged to treat common red blood cell disorders.

DOI: https://doi.org/10.1126/science.adh9215