bims-scepro 2024-07-07 papers

bims-scepro

Biomed News

on Stem cell proteostasis

Issue of 2024–07–07
thirty-six papers selected by
William Grey, University of York

Isolation of Human Hematopoietic Stem Cells from an Apheresis Sample.
Ex Vivo Evaluation of the Function of Hematopoietic Stem Cells in Toxicology of Metals.
LSD1/KDM1A and GFI1B repress endothelial fate and induce hematopoietic fate in induced pluripotent stem cell-derived hemogenic endotheliums.
Effect of Post-transplant Dietary Restriction on Hematopoietic Reconstitution and Maintenance of Reconstitution Capacity of Hematopoietic Stem Cells.
Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing.
Alkynyl nicotinamides show antileukemic activity in drug-resistant acute myeloid leukemia.
Nynrin preserves hematopoietic stem cell function by inhibiting the mitochondrial permeability transition pore opening.
Genetic deletion of JAM-C in pre-leukemic cells rewires leukemic stem cell gene expression program in AML.
SLPI overexpression in hMSCs could be implicated in the HSC gene expression profile in AML.
Second bone marrow transplantation into regenerating hematopoiesis enhances reconstitution of immune system.
DCAF15 control of cohesin dynamics sustains acute myeloid leukemia.
A novel LGALS1-depended and immune-associated fatty acid metabolism risk model in acute myeloid leukemia stem cells.
DNMT3A-R882: a mutation with many paradoxes.
CNS-wide repopulation by hematopoietic-derived microglia-like cells corrects progranulin deficiency in mice.
Pro-survival signaling regulates lipophagy essential for multiple myeloma resistance to stress-induced death.
Histone demethylation tones down leukemia through innate immunity.
Differential signalling requirements for RIPK1-dependent pyroptosis in neutrophils and macrophages.
The heterogeneity of erythroid cells: insight at the single-cell transcriptome level.
Activation of non-classical Wnt signaling pathway effectively enhances HLA-A presentation in acute myeloid leukemia.
Lysosomal cation channel TRPML1 suppression sensitizes acute myeloid leukemia cells to chemotherapeutics by inhibiting autophagy.
Exploring the potential of predicted miRNAs on the genes involved in the expansion of hematopoietic stem cells.
Intrinsic signaling pathways modulate targeted protein degradation.
NAD metabolism-related genes provide prognostic value and potential therapeutic insights for acute myeloid leukemia.
Molecular Modeling of Single- and Double-Hydrocarbon-Stapled Coiled-Coil Inhibitors against Bcr-Abl: Toward a Treatment Strategy for CML.
Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing.
The Interplay of Acetylation and Ubiquitination Controls PRMT1 Homeostasis.
Single-nucleus proteomics identifies regulators of protein transport.
Identification of De Novo Dividing Stem Cells.
Hematopoietic stem cell gene therapy for the treatment of SYNGAP1-related non-specific intellectual disability.
UPS-dependent strategies of protein quality control degradation.
Exploiting the potential of the ubiquitin-proteasome system in overcoming tyrosine kinase inhibitor resistance in chronic myeloid leukemia.
Engineered matrices reveal stiffness-mediated chemoresistance in patient-derived pancreatic cancer organoids.
Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum.
Establishment of a humanized mouse model using steady-state peripheral blood-derived hematopoietic stem and progenitor cells facilitates screening of cancer-targeted T-cell repertoires.
Protein modification and degradation in ferroptosis.
Aberrant early hematopoietic progenitor formation marks the onset of hematopoietic defects in Shwachman-Diamond syndrome.

Methods Mol Biol. 2024 Jul 06.

Isolation of Human Hematopoietic Stem Cells from an Apheresis Sample.

Pınar Siyah, Fatih Kocabaş.

  The hematopoietic system constantly produces new blood cells through hematopoiesis, and maintaining this balance is vital for human health. This balance is maintained by self-renewing hematopoietic stem cells (HSCs) and various progenitor cells. Under typical circumstances, HSCs are not abundantly found in peripheral blood; hence, their mobilization from the bone marrow is vital. Hematopoietic growth factors achieve this effectively, enabling mobilization and thus allowing blood sample and thus HSC collection via apheresis. Securing a sufficient supply of HSCs is vital for successful hematopoietic reconstitution and the rapid integration of committed cells. Thus, isolation and expansion of HSCs are crucial for convenient extraction, production of transplantable quantities, genetic modifications for enhanced therapeutic efficacy, and as a source of increased/expanded/synthesized blood cells in vitro. In conclusion, the isolation and expansion of HSCs play pivotal roles in both regenerative medicine and hematology. This protocol describes the isolation of human HSCs by providing an overview of the primary method for isolating human hematopoietic stem cells from apheresis blood samples and sheds light on human HSC studies and developments in research and medicine.

Keywords:  Apheresis; HSC isolation; Hematopoiesis; Hematopoietic stem cell transplantation (HSCT); Hematopoietic stem cells; Peripheral blood; Stem cell expansion

DOI:  https://doi.org/10.1007/7651_2024_557
Curr Protoc. 2024 Jul;4(7): e1038

Ex Vivo Evaluation of the Function of Hematopoietic Stem Cells in Toxicology of Metals.

Jiaojiao Wu, Ting Liu, Mengke Tang, Yalin Liu, Wei Wang, Chuanxuan Wang, Yingzi Ju, Yifan Zhao, Yubin Zhang.

  A variety of metals, e.g., lead (Pb), cadmium (Cd), and lithium (Li), are in the environment and are toxic to humans. Hematopoietic stem cells (HSCs) reside at the apex of hematopoiesis and are capable of generating all kinds of blood cells and self-renew to maintain the HSC pool. HSCs are sensitive to environmental stimuli. Metals may influence the function of HSCs by directly acting on HSCs or indirectly by affecting the surrounding microenvironment for HSCs in the bone marrow (BM) or niche, including cellular and extracellular components. Investigating the impact of direct and/or indirect actions of metals on HSCs contributes to the understanding of immunological and hematopoietic toxicology of metals. Treatment of HSCs with metals ex vivo, and the ensuing HSC transplantation assays, are useful for evaluating the impacts of the direct actions of metals on the function of HSCs. Investigating the mechanisms involved, given the rarity of HSCs, methods that require large numbers of cells are not suitable for signal screening; however, flow cytometry is a useful tool for signal screening HSCs. After targeting signaling pathways, interventions ex vivo and HSCs transplantation are required to confirm the roles of the signaling pathways in regulating the function of HSCs exposed to metals. Here, we describe protocols to evaluate the mechanisms of direct and indirect action of metals on HSCs. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Identify the impact of a metal on the competence of HSCs Basic Protocol 2: Identify the impact of a metal on the lineage bias of HSC differentiation Basic Protocol 3: Screen the potential signaling molecules in HSCs during metal exposure Alternate Protocol 1: Ex vivo treatment with a metal on purified HSCs Alternate Protocol 2: Ex vivo intervention of the signaling pathway regulating the function of HSCs during metal exposure.

Keywords:  competence; ex vivo intervention; hematopoietic stem cells; lineage bias; metal; signal screening

DOI:  https://doi.org/10.1002/cpz1.1038
Haematologica. 2024 Jul 04.

LSD1/KDM1A and GFI1B repress endothelial fate and induce hematopoietic fate in induced pluripotent stem cell-derived hemogenic endotheliums.

Huan Zhang, Marten Hansen, Franca Di Summa, Marieke Von Lindern, Nynke Gillemans, Wilfred F J Van IJcken, Arthur Flohr Svendsen, Sjaak Philipsen, Bert Van der Reijden, Eszter Varga, Emile Van den Akker.

Differentiation of induced pluripotent stem cells (iPSCs) into hematopoietic lineages offers great therapeutic potential. During embryogenesis, hemogenic endothelium (HE) gives rise to hematopoietic stem and progenitor cells through the endothelial-to-hematopoietic transition (EHT). Understanding this process using iPSCs is key to generating functional hematopoietic stem cells (HSCs), a currently unmet challenge. In this study, we examined the role of the transcriptional factor GFI1B and its co-factor LSD1/KDM1A in EHT. To this end, we employed patient-derived iPSC lines with a dominant negative dysfunctional GFI1BQ287* and irreversible pharmacological LSD1/KDM1A inhibition in healthy iPSC lines. The formation of HE remained unaffected; however, hematopoietic output was severely reduced in both conditions. Single-cell RNA sequencing (scRNAseq) performed on the CD144+/CD31+ population derived from healthy iPSCs revealed similar expression dynamics of genes associated with in vivo EHT. Interestingly, LSD1/KDM1A inhibition in healthy lines before EHT resulted in a complete absence of hematopoietic output. However, uncommitted HE cells did not display GFI1B expression, suggesting a timed transcriptional program. To test this hypothesis, we ectopically expressed GFI1B in uncommitted HE cells, leading to downregulation of endothelial genes and upregulation of hematopoietic genes, including GATA2, KIT, RUNX1, and SPI1. Thus, we demonstrate that LSD1/KDM1A and GFI1B can function at distinct temporal points in different cellular subsets during EHT. Although GFI1B is not detected in uncommitted HE cells, its ectopic expression allows for partial hematopoietic specification. These data indicate that precisely timed expression of specific transcriptional regulators during EHT is crucial to the eventual outcome of EHT.

DOI: https://doi.org/10.3324/haematol.2024.285214
Stem Cell Rev Rep. 2024 Jul 05.

Effect of Post-transplant Dietary Restriction on Hematopoietic Reconstitution and Maintenance of Reconstitution Capacity of Hematopoietic Stem Cells.

Si Tao, Xingxing Qiu, Yiting Wang, Rongrong Qiu, Chenghui Yu, Man Sun, Lulu Liu, Zhendong Tao, Liu Zhang, Duozhuang Tang.

  Hematopoietic cell transplantation (HCT) is an important therapy for many hematological malignancies as well as some non-malignant diseases. Post-transplant hematopoiesis is affected by multiple factors, and the mechanisms of delayed post-transplant hematopoiesis remain poorly understood. Patients undergoing HCT often suffer from significantly reduced food intake due to complications induced by preconditioning treatments. Here, we used a dietary restriction (DR) mouse model to study the effect of post-transplant dietary reduction on hematopoiesis and hematopoietic stem cells (HSCs). We found that post-transplant DR significantly inhibited both lymphopoiesis and myelopoiesis in the primary recipient mice. However, when bone marrow cells (BMCs) from the primary recipient mice were serially transplanted into secondary and tertiary recipient mice, the HSCs derived from the primary recipient mice, which were exposed to post-transplant DR, exhibited a much higher reconstitution capacity. Transplantation experiments with purified HSCs showed that post-transplant DR greatly inhibited hematopoietic stem cell (HSC) expansion. Additionally, post-transplant DR reshaped the gut microbiotas of the recipient mice, which inhibited inflammatory responses and thus may have contributed to maintaining HSC function. Our findings may have important implications for clinical work because reduced food intake and problems with digestion and absorption are common in patients undergoing HCT.

Keywords:  Dietary restriction; Hematopoiesis; Post-transplant

DOI:  https://doi.org/10.1007/s12015-024-10754-y
Bio Protoc. 2024 Jun 20. 14(12): e5020

Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing.

Hongzhe Li, Sandro Bräunig, Stefan Scheding.

  The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche. Key features • Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq • Targeted strategy for enriching low-frequency stromal cells.

Keywords:  Bone marrow stromal cells; CD235a; CD271; CD45; Flow cytometry; Human bone marrow microenvironment; Non-hematopoietic cells

DOI:  https://doi.org/10.21769/BioProtoc.5020
J Clin Invest. 2024 Jun 17. pii: e169245. [Epub ahead of print]134(12):

Alkynyl nicotinamides show antileukemic activity in drug-resistant acute myeloid leukemia.

Baskar Ramdas, Neetu Dayal, Ruchi Pandey, Elizabeth Larocque, Rahul Kanumuri, Santhosh Kumar Pasupuleti, Sheng Liu, Chrysi Kanellopoulou, Elizabeth Fei Yin Chu, Rodrigo Mohallem, Saniya Virani, Gaurav Chopra, Uma K Aryal, Rena Lapidus, Jun Wan, Ashkan Emadi, Laura S Haneline, Frederick W Holtsberg, M Javad Aman, Herman O Sintim, Reuben Kapur.

  Activating mutations of FLT3 contribute to deregulated hematopoietic stem and progenitor cell (HSC/Ps) growth and survival in patients with acute myeloid leukemia (AML), leading to poor overall survival. AML patients treated with investigational drugs targeting mutant FLT3, including Quizartinib and Crenolanib, develop resistance to these drugs. Development of resistance is largely due to acquisition of cooccurring mutations and activation of additional survival pathways, as well as emergence of additional FLT3 mutations. Despite the high prevalence of FLT3 mutations and their clinical significance in AML, there are few targeted therapeutic options available. We have identified 2 novel nicotinamide-based FLT3 inhibitors (HSN608 and HSN748) that target FLT3 mutations at subnanomolar concentrations and are potently effective against drug-resistant secondary mutations of FLT3. These compounds show antileukemic activity against FLT3ITD in drug-resistant AML, relapsed/refractory AML, and in AML bearing a combination of epigenetic mutations of TET2 along with FLT3ITD. We demonstrate that HSN748 outperformed the FDA-approved FLT3 inhibitor Gilteritinib in terms of inhibitory activity against FLT3ITD in vivo.

Keywords:  Hematology; Hematopoietic stem cells; Stem cells

DOI:  https://doi.org/10.1172/JCI169245
Cell Stem Cell. 2024 Jun 25. pii: S1934-5909(24)00215-7. [Epub ahead of print]

Nynrin preserves hematopoietic stem cell function by inhibiting the mitochondrial permeability transition pore opening.

Chengfang Zhou, Mei Kuang, Yin Tao, Jianming Wang, Yu Luo, Yinghao Fu, Zhe Chen, Yuanyuan Liu, Zhigang Li, Weiru Wu, Li Wang, Ying Dou, Junping Wang, Yu Hou.

  Mitochondria are key regulators of hematopoietic stem cell (HSC) homeostasis. Our research identifies the transcription factor Nynrin as a crucial regulator of HSC maintenance by modulating mitochondrial function. Nynrin is highly expressed in HSCs under both steady-state and stress conditions. The knockout Nynrin diminishes HSC frequency, dormancy, and self-renewal, with increased mitochondrial dysfunction indicated by abnormal mPTP opening, mitochondrial swelling, and elevated ROS levels. These changes reduce HSC radiation tolerance and promote necrosis-like phenotypes. By contrast, Nynrin overexpression in HSCs diminishes irradiation (IR)-induced lethality. The deletion of Nynrin activates Ppif, leading to overexpression of cyclophilin D (CypD) and further mitochondrial dysfunction. Strategies such as Ppif haploinsufficiency or pharmacological inhibition of CypD significantly mitigate these effects, restoring HSC function in Nynrin-deficient mice. This study identifies Nynrin as a critical regulator of mitochondrial function in HSCs, highlighting potential therapeutic targets for preserving stem cell viability during cancer treatment.

Keywords:  Nynrin; Ppif; ROS; hematopoietic stem cells; irradiation; mPTP opening; mitochondrial membrane potential; mitochondrial metabolism; mitochondrial swelling; radiotherapy

DOI:  https://doi.org/10.1016/j.stem.2024.06.007
Blood Adv. 2024 Jul 02. pii: bloodadvances.2023011747. [Epub ahead of print]

Genetic deletion of JAM-C in pre-leukemic cells rewires leukemic stem cell gene expression program in AML.

Julien M P Grenier, Céline Testut, Matthieu Bal, Florence Bardin, Maria De Grandis, Véronique Gelsi-Boyer, Julien Vernerny, Marjorie C Delahaye, Samuel Granjeaud, Christophe Zemmour, Jean-François Spinella, Triantafyllos Chavakis, Stephane Jc Mancini, Jean-Marie Boher, Josée Hébert, Guy Sauvageau, Norbert Vey, Juerg Schwaller, Marie-Anne Hospital, Cyril Fauriat, Michel A Aurrand-Lions.

The leukemic stem cell (LSC) score LSC-17 based on a stemness-related gene expression signature is an indicator of poor disease outcome in acute myeloid leukemia (AML). However, our understanding of the relationships between LSC and pre-leukemic cells is still incomplete. In particular, it is not known whether "niche-anchoring" of pre-leukemic cell affects disease evolution. To address this issue, we conditionally inactivated the adhesion molecule JAM-C expressed by haematopoietic stem cells (HSC) and LSC in an inducible iMLL-AF9-driven AML mouse model. Deletion of Jam3 (encoding JAM-C) before induction of the leukemia-initiating iMLL-AF9 fusion resulted in a shift from long term to short term-HSC expansion, without affecting disease initiation and progression. In vitro experiments showed that JAM-C controlled leukemic cell nesting irrespective of the bone marrow stromal cells used. RNA sequencing performed on leukemic HSC isolated from diseased mice revealed that genes upregulated in Jam3-deficient animals belonged to Activation Protein-1 (AP-1) and TNF-/NFB pathways. Human orthologs of dysregulated genes allowed to identify a score based on AP-1/TNF-a gene expression that was distinct and complementary from LSC-17 score. Sub-stratification of AML patients with LSC-17 and AP-1/TNF-genes signature defined four groups with median survival ranging from below one year to a median not reached after 8 years. Finally, coculture experiments showed that AP-1 activation in leukemic cells was dependent on the nature of stromal cells. Altogether, our results identify the AP-1/TNF- gene signature as a proxy of LSC anchoring in specific bone marrow niches which improves the prognosis value of the LSC-17 score. NCT02320656.

DOI: https://doi.org/10.1182/bloodadvances.2023011747
Sci Rep. 2024 Jul 05. 14(1): 15550

SLPI overexpression in hMSCs could be implicated in the HSC gene expression profile in AML.

Pedro L Azevedo, Simone Maradei, Ricardo de Sá Bigni, Jordana Santos Ramires Aragao, Eliana Abdelhay, Renata Binato.

Acute myeloid leukaemia (AML) is a severe haematological neoplasm that originates from the transformation of haematopoietic stem cells (HSCs) into leukaemic stem cells (LSCs). The bone marrow (BM) microenvironment, particularly that of mesenchymal stromal cells (hMSCs), plays a crucial role in the maintenance of HSCs. In this context, we explored whether alterations in the secretome of hMSCs derived from AML patients (hMSC-AML) could impact HSC gene expression. Proteomic analysis revealed that the secretome of coculture assays with hMSC-AMLs and HSC from healthy donor is altered, with increased levels of secretory leukocyte protease inhibitor (SLPI), a protein associated with important processes for maintenance of the haematopoietic niche that has already been described to be altered in several tumours. Increased SLPI expression was also observed in the BM plasma of AML patients. Transcriptome analysis of HSCs cocultured with hMSC-AML in comparison with HSCs cocultured with hMSC-HD revealed altered expression of SLPI target genes associated with the cell cycle, proliferation, and apoptosis. Important changes were identified, such as increased expression levels of CCNA2, CCNE2, CCND2, CD133 and CDK1 and decreased levels of CDKN2A and IGFBP3, among others. Overall, these findings suggest that the altered secretome of coculture assays with hMSC-AMLs and HSC from healthy donor, particularly increased SLPI expression, can contribute to gene expression changes in HSCs, potentially influencing important molecular mechanisms related to AML development and progression.

DOI: https://doi.org/10.1038/s41598-024-66400-7
Front Immunol. 2024 ;15 1405210

Second bone marrow transplantation into regenerating hematopoiesis enhances reconstitution of immune system.

Kateřina Faltusová, Martin Báječný, Tomáš Heizer, Petr Páral, Chia-Ling Chen, Katarína Szikszai, Pavel Klener, Emanuel Nečas.

  In bone marrow transplantation (BMT), hematopoiesis-reconstituting cells are introduced following myeloablative treatment, which eradicates existing hematopoietic cells and disrupts stroma within the hematopoietic tissue. Both hematopoietic cells and stroma then undergo regeneration. Our study compares the outcomes of a second BMT administered to mice shortly after myeloablative treatment and the first BMT, with those of a second BMT administered to mice experiencing robust hematopoietic regeneration after the initial transplant. We evaluated the efficacy of the second BMT in terms of engraftment efficiency, types of generated blood cells, and longevity of function. Our findings show that regenerating hematopoiesis readily accommodates newly transplanted stem cells, including those endowed with a robust capacity for generating B and T cells. Importantly, our investigation uncovered a window for preferential engraftment of transplanted stem cells coinciding with the resumption of blood cell production. Repeated BMT could intensify hematopoiesis reconstitution and enable therapeutic administration of genetically modified autologous stem cells.

Keywords:  B cells; T cells; hematopoiesis; immune system; regeneration; stem cell; transplantation

DOI:  https://doi.org/10.3389/fimmu.2024.1405210
Nat Commun. 2024 Jul 03. 15(1): 5604

DCAF15 control of cohesin dynamics sustains acute myeloid leukemia.

Grant P Grothusen, Renxu Chang, Zhendong Cao, Nan Zhou, Monika Mittal, Arindam Datta, Phillip Wulfridge, Thomas Beer, Baiyun Wang, Ning Zheng, Hsin-Yao Tang, Kavitha Sarma, Roger A Greenberg, Junwei Shi, Luca Busino.

The CRL4-DCAF15 E3 ubiquitin ligase complex is targeted by the aryl-sulfonamide molecular glues, leading to neo-substrate recruitment, ubiquitination, and proteasomal degradation. However, the physiological function of DCAF15 remains unknown. Using a domain-focused genetic screening approach, we reveal DCAF15 as an acute myeloid leukemia (AML)-biased dependency. Loss of DCAF15 results in suppression of AML through compromised replication fork integrity and consequent accumulation of DNA damage. Accordingly, DCAF15 loss sensitizes AML to replication stress-inducing therapeutics. Mechanistically, we discover that DCAF15 directly interacts with the SMC1A protein of the cohesin complex and destabilizes the cohesin regulatory factors PDS5A and CDCA5. Loss of PDS5A and CDCA5 removal precludes cohesin acetylation on chromatin, resulting in uncontrolled chromatin loop extrusion, defective DNA replication, and apoptosis. Collectively, our findings uncover an endogenous, cell autonomous function of DCAF15 in sustaining AML proliferation through post-translational control of cohesin dynamics.

DOI: https://doi.org/10.1038/s41467-024-49882-x
Cell Death Dis. 2024 Jul 05. 15(7): 482

A novel LGALS1-depended and immune-associated fatty acid metabolism risk model in acute myeloid leukemia stem cells.

Huanhuan Qin, Meixi Peng, Jingsong Cheng, Zhenyu Wang, Yinghui Cui, Yongxiu Huang, Yaoqi Gui, Yanni Sun, Wenqiong Xiang, Xiaomei Huang, Ting Huang, Li Wang, Jieping Chen, Yu Hou.

Leukemia stem cells (LSCs) are recognized as the root cause of leukemia initiation, relapse, and drug resistance. Lipid species are highly abundant and essential component of human cells, which often changed in tumor microenvironment. LSCs remodel lipid metabolism to sustain the stemness. However, there is no useful lipid related biomarker has been approved for clinical practice in AML prediction and treatment. Here, we constructed and verified fatty acid metabolism-related risk score (LFMRS) model based on TCGA database via a series of bioinformatics analysis, univariate COX regression analysis, and multivariate COX regression analysis, and found that the LFMRS model could be an independent risk factor and predict the survival time of AML patients combined with age. Moreover, we revealed that Galectin-1 (LGALS1, the key gene of LFMRS) was highly expressed in LSCs and associated with poor prognosis of AML patients, and LGALS1 repression inhibited AML cell and LSC proliferation, enhanced cell apoptosis, and decreased lipid accumulation in vitro. LGALS1 repression curbed AML progression, lipid accumulation, and CD8+ T and NK cell counts in vivo. Our study sheds light on the roles of LFMRS (especially LGALS1) model in AML, and provides information that may help clinicians improve patient prognosis and develop personalized treatment regimens for AML.

DOI: https://doi.org/10.1038/s41419-024-06865-6
Ann Hematol. 2024 Jul 06.

DNMT3A-R882: a mutation with many paradoxes.

Pourya Arbab Jafari, Ramin Bagheri, Soroush Lavasani, Sajad Goudarzi.

  Understanding the underlying mechanism of acute myeloid leukemia (AML) has led to the discovery of novel biomarkers to help predict, treat and monitor leukemia. DNA (cytosine-5)-methyltransferase 3 A (DNMT3A) is considered a prognostic and therapeutic epigenetic target in AML patients with a hotspot mutation of R882. R882 mutation is associated with impaired differentiation of Hematopoietic stem cells in the bone marrow and disease progression. The prevalence of R882 mutation varied in different ethnicities and countries, and similarly, its prognostic impact differed among numerous studies. Nevertheless, the co-occurrence of mutations in R882 with NPM1 and FLT3 has been reported more frequently and is associated with a worse prognosis. These studies also suggest diverse results regarding bone marrow transplantation response as a treatment, while chemoresistance is reached as a conclusive outcome These findings highlight the crucial need for an in-depth discussion on the significance of the R882 mutation in AML patients. Understanding its impact on leukemic transformation, prognosis, and treatment is vital for advancing clinical implications.

Keywords:  AML; DNMT3A; Prognosis; R882 mutation

DOI:  https://doi.org/10.1007/s00277-024-05874-x
Nat Commun. 2024 Jul 05. 15(1): 5654

CNS-wide repopulation by hematopoietic-derived microglia-like cells corrects progranulin deficiency in mice.

Pasqualina Colella, Ruhi Sayana, Maria Valentina Suarez-Nieto, Jolanda Sarno, Kwamina Nyame, Jian Xiong, Luisa Natalia Pimentel Vera, Jessica Arozqueta Basurto, Marco Corbo, Anay Limaye, Kara L Davis, Monther Abu-Remaileh, Natalia Gomez-Ospina.

Hematopoietic stem cell transplantation can deliver therapeutic proteins to the central nervous system (CNS) through transplant-derived microglia-like cells. However, current conditioning approaches result in low and slow engraftment of transplanted cells in the CNS. Here we optimized a brain conditioning regimen that leads to rapid, robust, and persistent microglia replacement without adverse effects on neurobehavior or hematopoiesis. This regimen combines busulfan myeloablation and six days of Colony-stimulating factor 1 receptor inhibitor PLX3397. Single-cell analyses revealed unappreciated heterogeneity of microglia-like cells with most cells expressing genes characteristic of homeostatic microglia, brain-border-associated macrophages, and unique markers. Cytokine analysis in the CNS showed transient inductions of myeloproliferative and chemoattractant cytokines that help repopulate the microglia niche. Bone marrow transplant of progranulin-deficient mice conditioned with busulfan and PLX3397 restored progranulin in the brain and eyes and normalized brain lipofuscin storage, proteostasis, and lipid metabolism. This study advances our understanding of CNS repopulation by hematopoietic-derived cells and demonstrates its therapeutic potential for treating progranulin-dependent neurodegeneration.

DOI: https://doi.org/10.1038/s41467-024-49908-4
Cell Rep. 2024 Jul 03. pii: S2211-1247(24)00774-5. [Epub ahead of print]43(7): 114445

Pro-survival signaling regulates lipophagy essential for multiple myeloma resistance to stress-induced death.

Peng Peng, Colin Chavel, Wensheng Liu, Louise M Carlson, Sha Cao, Adam Utley, Scott H Olejniczak, Kelvin P Lee.

  Pro-survival metabolic adaptations to stress in tumorigenesis remain less well defined. We find that multiple myeloma (MM) is unexpectedly dependent on beta-oxidation of long-chain fatty acids (FAs) for survival under both basal and stress conditions. However, under stress conditions, a second pro-survival signal is required to sustain FA oxidation (FAO). We previously found that CD28 is expressed on MM cells and transduces a significant pro-survival/chemotherapy resistance signal. We now find that CD28 signaling regulates autophagy/lipophagy that involves activation of the Ca2+→AMPK→ULK1 axis and regulates the translation of ATG5 through HuR, resulting in sustained lipophagy, increased FAO, and enhanced MM survival. Conversely, blocking autophagy/lipophagy sensitizes MM to chemotherapy in vivo. Our findings link a pro-survival signal to FA availability needed to sustain the FAO required for cancer cell survival under stress conditions and identify lipophagy as a therapeutic target to overcome treatment resistance in MM.

Keywords:  CD28; CP: Cancer; CP: Metabolism; autophagy; fatty acid metabolism; fatty acid oxidation; lipid droplets; lipophagy; multiple myeloma; pro-survival regulation

DOI:  https://doi.org/10.1016/j.celrep.2024.114445
Sci Immunol. 2024 Jul 05. 9(97): eadr2967

Histone demethylation tones down leukemia through innate immunity.

Andre Monteleone, Gabriel K Griffin.

Histone demethylation by PHF8 initiates innate immune signaling in acute myeloid leukemia, elucidating a novel therapeutic strategy.

DOI: https://doi.org/10.1126/sciimmunol.adr2967
Cell Death Dis. 2024 Jul 04. 15(7): 479

Differential signalling requirements for RIPK1-dependent pyroptosis in neutrophils and macrophages.

See Jie Yow, Safwah Nasuha Rosli, Paul E Hutchinson, Kaiwen W Chen.

TLR4 and TNFR1 signalling promotes potent proinflammatory signal transduction events, thus, are often hijacked by pathogenic microorganisms. We recently reported that myeloid cells retaliate Yersinia blockade of TAK1/IKK signalling by triggering RIPK1-dependent caspase-8 activation that promotes downstream GSDMD and GSDME-mediated pyroptosis in macrophages and neutrophils respectively. However, the upstream signalling events for RIPK1 activation in these cells are not well defined. Here, we demonstrate that unlike in macrophages, RIPK1-driven pyroptosis and cytokine priming in neutrophils are driven through TNFR1 signalling, while TLR4-TRIF signalling is dispensable. Furthermore, we demonstrate that activation of RIPK1-dependent pyroptosis in neutrophils during Yersinia infection requires IFN-γ priming, which serves to induce surface TNFR1 expression and amplify soluble TNF secretion. In contrast, macrophages utilise both TNFR1 and TLR4-TRIF signalling to trigger cell death, but only require TRIF but not autocrine TNFR1 for cytokine production. Together, these data highlight the emerging theme of cell type-specific regulation in cell death and immune signalling in myeloid cells.

DOI: https://doi.org/10.1038/s41419-024-06871-8
Cell Tissue Res. 2024 Jul 02.

The heterogeneity of erythroid cells: insight at the single-cell transcriptome level.

Jingwei Wang, Yipeng Liang, Changlu Xu, Jie Gao, Jingyuan Tong, Lihong Shi.

  Erythroid cells, the most prevalent cell type in blood, are one of the earliest products and permeate through the entire process of hematopoietic development in the human body, the oxygen-transporting function of which is crucial for maintaining overall health and life support. Previous investigations into erythrocyte differentiation and development have primarily focused on population-level analyses, lacking the single-cell perspective essential for comprehending the intricate pathways of erythroid maturation, differentiation, and the encompassing cellular heterogeneity. The continuous optimization of single-cell transcriptome sequencing technology, or single-cell RNA sequencing (scRNA-seq), provides a powerful tool for life sciences research, which has a particular superiority in the identification of unprecedented cell subgroups, the analyzing of cellular heterogeneity, and the transcriptomic characteristics of individual cells. Over the past decade, remarkable strides have been taken in the realm of single-cell RNA sequencing technology, profoundly enhancing our understanding of erythroid cells. In this review, we systematically summarize the recent developments in single-cell transcriptome sequencing technology and emphasize their substantial impact on the study of erythroid cells, highlighting their contributions, including the exploration of functional heterogeneity within erythroid populations, the identification of novel erythrocyte subgroups, the tracking of different erythroid lineages, and the unveiling of mechanisms governing erythroid fate decisions. These findings not only invigorate erythroid cell research but also offer new perspectives on the management of diseases related to erythroid cells.

Keywords:  Cell heterogeneity; Erythroid cells; Erythropoiesis; Single-cell transcriptome sequencing; Subpopulations

DOI:  https://doi.org/10.1007/s00441-024-03903-9
Front Oncol. 2024 ;14 1336106

Activation of non-classical Wnt signaling pathway effectively enhances HLA-A presentation in acute myeloid leukemia.

YuHan Ma, JunShuai Yue, Ling Gao, JingXin Zhou, Wei Chen, Jing Su, JinRong Yao, QiaoMei Shi, XiaoDong Zhao, Na Hu.

   Objective: The escape from T cell-mediated immune surveillance is an important cause of death for patients with acute myeloid leukemia (AML). This study aims to identify clonal heterogeneity in leukemia progenitor cells and explore molecular or signaling pathways associated with AML immune escape.
Methods: Single-cell RNA sequencing (scRNA-seq) was performed to identified AML-related cellular subsets, and intercellular communication was analyzed to investigate molecular mechanisms associated with AML immune escape. Bulk RNA sequencing (RNA-seq) was performed to screen differentially expressed genes (DEGs) related to hematopoietic stem cell progenitors (HSC-Prog) in AML, and critical ore signaling pathways and hub genes were found by Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The mRNA level of the hub gene was verified using quantitative real-time PCR (qRT-PCR) and the protein level of human leukocyte antigen A (HLA-A) using enzyme-linked immuno sorbent assay (ELISA).
Results: scRNA-seq analysis revealed a large heterogeneity of HSC-Prog across samples, and the intercellular communication analysis indicated a strong association between HSC-Prog and CD8+-T cells, and HSC-Prog also had an association with HLA-A. Transcriptome analysis identified 1748 DEGs, enrichment analysis results showed that non-classical wnt signaling pathway was associated with AML, and 4 pathway-related genes (RHOA, RYK, CSNK1D, NLK) were obtained. After qRT-PCR and ELISA validation, hub genes and HLA-A were found to be down-regulated in AML and up-regulated after activation of the non-classical Wnt signaling pathway.
Conclusion: In this study, clonal heterogeneity of HSC-Prog cells in AML was identified, non-classical wnt signaling pathways associated with AML were identified, and it was verified that HLA-A could be upregulated by activation of non-classical wnt signaling, thereby increasing antigen presentation.

Keywords:  HLA-A; acute myeloid leukemia; immune escape; non-classical Wnt signaling pathway; scRNA-seq

DOI:  https://doi.org/10.3389/fonc.2024.1336106
Mol Cell Biochem. 2024 Jun 29.

Lysosomal cation channel TRPML1 suppression sensitizes acute myeloid leukemia cells to chemotherapeutics by inhibiting autophagy.

Meifang Dai, Bingqian Lin, Hao Li, Youming Wang, Miaomiao Wu, Yanan Wei, Wenping Zeng, Lili Qu, Chunlei Cang, Xingbing Wang.

  Despite the implementation of novel therapeutic regimens and extensive research efforts, chemoresistance remains a formidable challenge in the treatment of acute myeloid leukemia (AML). Notably, the involvement of lysosomes in chemoresistance has sparked interest in developing lysosome-targeted therapies to sensitize tumor cells to currently approved chemotherapy or as innovative pharmacological approaches. Moreover, as ion channels on the lysosomal membrane are critical regulators of lysosomal function, they present potential as novel targets for enhancing chemosensitivity. Here, we discovered that the expression of a lysosomal cation channel, namely transient receptor potential mucolipin 1 (TRPML1), was elevated in AML cells. Inhibiting TRPML1 individually does not impact the proliferation and apoptosis of AML cells. Importantly, inhibition of TRPML1 demonstrated the potential to modulate the sensitivity of AML cells to chemotherapeutic agents. Exploration of the underlying mechanisms revealed that suppression of TRPML1 impaired autophagy while concurrently increasing the production of reactive oxygen species (ROS) and ROS-mediated lipid peroxidation (Lipid-ROS) in AML cells. Finally, the knockdown of TRPML1 significantly reduced OCI-AML3 tumor growth following chemotherapy in a mouse model of human leukemia. In summary, targeting TRPML1 represents a promising approach for combination therapy aimed at enhancing chemosensitivity in treating AML.

Keywords:  Acute myeloid leukemia; Autophagy; Chemosensitivity; Lysosome; TRPML1

DOI:  https://doi.org/10.1007/s11010-024-05054-5
Sci Rep. 2024 Jul 05. 14(1): 15551

Exploring the potential of predicted miRNAs on the genes involved in the expansion of hematopoietic stem cells.

Mohammad Elahimanesh, Nafiseh Shokri, Ronak Shabani, Maryam Rahimi, Mohammad Najafi.

  A major challenge in therapeutic approaches applying hematopoietic stem cells (HSCs) is the cell quantity. The primary objective of this study was to predict the miRNAs and anti-miRNAs using bioinformatics tools and investigate their effects on the expression levels of key genes predicted in the improvement of proliferation, and the inhibition of differentiation in HSCs isolated from Human umbilical cord blood (HUCB). A network including genes related to the differentiation and proliferation stages of HSCs was constructed by enriching data of text (PubMed) and StemChecker server with KEGG signaling pathways, and was improved using GEO datasets. Bioinformatics tools predicted a profile from miRNAs containing miR-20a-5p, miR-423-5p, and chimeric anti-miRNA constructed from 5'-miR-340/3'-miR-524 for the high-score genes (RB1, SMAD4, STAT1, CALML4, GNG13, and CDKN1A/CDKN1B genes) in the network. The miRNAs and anti-miRNA were transferred into HSCs using polyethylenimine (PEI). The gene expression levels were estimated using the RT-qPCR technique in the PEI + (miRNA/anti-miRNA)-contained cell groups (n = 6). Furthermore, CD markers (90, 16, and 45) were evaluated using flow cytometry. Strong relationships were found between the high-score genes, miRNAs, and chimeric anti-miRNA. The RB1, SMAD4, and STAT1 gene expression levels were decreased by miR-20a-5p (P < 0.05). Additionally, the anti-miRNA increased the gene expression level of GNG13 (P < 0.05), whereas the miR-423-5p decreased the CDKN1A gene expression level (P < 0.01). The cellular count also increased significantly (P < 0.05) but the CD45 differentiation marker did not change in the cell groups. The study revealed the predicted miRNA/anti-miRNA profile expands HSCs isolated from HUCB. While miR-20a-5p suppressed the RB1, SMAD4, and STAT1 genes involved in cellular differentiation, the anti-miRNA promoted the GNG13 gene related to the proliferation process. Notably, the mixed miRNA/anti-miRNA group exhibited the highest cellular expansion. This approach could hold promise for enhancing the cell quantity in HSC therapy.

Keywords:  Cord blood; Differentiation; Gene network; Hematopoietic stem cells; Proliferation; miRNA

DOI:  https://doi.org/10.1038/s41598-024-66614-9
Nat Commun. 2024 Jul 02. 15(1): 5379

Intrinsic signaling pathways modulate targeted protein degradation.

Yuki Mori, Yoshino Akizuki, Rikuto Honda, Miyu Takao, Ayaka Tsuchimoto, Sota Hashimoto, Hiroaki Iio, Masakazu Kato, Ai Kaiho-Soma, Yasushi Saeki, Jun Hamazaki, Shigeo Murata, Toshikazu Ushijima, Naoko Hattori, Fumiaki Ohtake.

Targeted protein degradation is a groundbreaking modality in drug discovery; however, the regulatory mechanisms are still not fully understood. Here, we identify cellular signaling pathways that modulate the targeted degradation of the anticancer target BRD4 and related neosubstrates BRD2/3 and CDK9 induced by CRL2VHL- or CRL4CRBN -based PROTACs. The chemicals identified as degradation enhancers include inhibitors of cellular signaling pathways such as poly-ADP ribosylation (PARG inhibitor PDD00017273), unfolded protein response (PERK inhibitor GSK2606414), and protein stabilization (HSP90 inhibitor luminespib). Mechanistically, PARG inhibition promotes TRIP12-mediated K29/K48-linked branched ubiquitylation of BRD4 by facilitating chromatin dissociation of BRD4 and formation of the BRD4-PROTAC-CRL2VHL ternary complex; by contrast, HSP90 inhibition promotes BRD4 degradation after the ubiquitylation step. Consequently, these signal inhibitors sensitize cells to the PROTAC-induced apoptosis. These results suggest that various cell-intrinsic signaling pathways spontaneously counteract chemically induced target degradation at multiple steps, which could be liberated by specific inhibitors.

DOI: https://doi.org/10.1038/s41467-024-49519-z
Front Immunol. 2024 ;15 1417398

NAD metabolism-related genes provide prognostic value and potential therapeutic insights for acute myeloid leukemia.

Yuncan Cao, Wenjing Shu, Peng Jin, Jianfeng Li, Hongming Zhu, Xinjie Chen, Yongmei Zhu, Xi Huang, Wenyan Cheng, Yang Shen.

   Introduction: Acute myeloid leukemia (AML) is an aggressive blood cancer with high heterogeneity and poor prognosis. Although the metabolic reprogramming of nicotinamide adenine dinucleotide (NAD) has been reported to play a pivotal role in the pathogenesis of acute myeloid leukemia (AML), the prognostic value of NAD metabolism and its correlation with the immune microenvironment in AML remains unclear.
Methods: We utilized our large-scale RNA-seq data on 655 patients with AML and the NAD metabolism-related genes to establish a prognostic NAD metabolism score based on the sparse regression analysis. The signature was validated across three independent datasets including a total of 1,215 AML patients. ssGSEA and ESTIMATE algorithms were employed to dissect the tumor immune microenvironment. Ex vivo drug screening and in vitro experimental validation were performed to identify potential therapeutic approaches for the high-risk patients. In vitro knockdown and functional experiments were employed to investigate the role of SLC25A51, a mitochondrial NAD+ transporter gene implicated in the signature.
Results: An 8-gene NAD metabolism signature (NADM8) was generated and demonstrated a robust prognostic value in more than 1,800 patients with AML. High NADM8 score could efficiently discriminate AML patients with adverse clinical characteristics and genetic lesions and serve as an independent factor predicting a poor prognosis. Immune microenvironment analysis revealed significant enrichment of distinct tumor-infiltrating immune cells and activation of immune checkpoints in patients with high NADM8 scores, acting as a potential biomarker for immune response evaluation in AML. Furthermore, ex vivo drug screening and in vitro experimental validation in a panel of 9 AML cell lines demonstrated that the patients with high NADM8 scores were more sensitive to the PI3K inhibitor, GDC-0914. Finally, functional experiments also substantiated the critical pathogenic role of the SLC25A51 in AML, which could be a promising therapeutic target.
Conclusion: Our study demonstrated that NAD metabolism-related signature can facilitate risk stratification and prognosis prediction in AML and guide therapeutic decisions including both immunotherapy and targeted therapies.

Keywords:  NAD metabolism; acute myeloid leukemia; immune microenvironment; prognostic signature; targeted therapy

DOI:  https://doi.org/10.3389/fimmu.2024.1417398
J Phys Chem B. 2024 Jul 01.

Molecular Modeling of Single- and Double-Hydrocarbon-Stapled Coiled-Coil Inhibitors against Bcr-Abl: Toward a Treatment Strategy for CML.

Maria Carolina P Lima, Braxten D Hornsby, Carol S Lim, Thomas E Cheatham.

The chimeric oncoprotein Bcr-Abl is the causative agent of virtually all chronic myeloid leukemias and a subset of acute lymphoblastic leukemias. As a result of the so-called Philadelphia chromosome translocation t(9;22), Bcr-Abl manifests as a constitutively active tyrosine kinase, which promotes leukemogenesis by activation of cell cycle signaling pathways. Constitutive and oncogenic activation is mediated by an N-terminal coiled-coil oligomerization domain in Bcr (Bcr-CC), presenting a therapeutic target for inhibition of Bcr-Abl activity toward the treatment of Bcr-Abl+ leukemias. Previously, we demonstrated that a rationally designed Bcr-CC mutant, CCmut3, exerts a dominant negative effect upon Bcr-Abl activity by preferential oligomerization with Bcr-CC. Moreover, we have shown that conjugation to a leukemia-specific cell-penetrating peptide (CPP-CCmut3) improves intracellular delivery and activity. However, our full-length CPP-CCmut3 construct (81 aa) is encumbered by an intrinsically high degree of conformational variability and susceptibility to proteolytic degradation relative to traditional small-molecule therapeutics. Here, we iterate a new generation of CCmut3 inhibitors against Bcr-CC-mediated Bcr-Abl assembly designed to address these constraints through incorporation of all-hydrocarbon staples spanning i and i + 7 positions in α-helix 2 (CPP-CCmut3-st). We utilize computational modeling and biomolecular simulation to evaluate single- and double-stapled CCmut3 candidates in silico for dynamics and binding energetics. We further model a truncated system characterized by the deletion of α-helix 1 and the flexible loop linker, which are known to impart high conformational variability. To study the impact of the N-terminal cyclic CPP toward model stability and inhibitor activity, we also model the full-length and truncated systems devoid of the CPP, with a cyclized CPP, and with an open-configuration CPP, for a total of six systems that comprise our library. From this library, we present lead-stapled peptide candidates to be synthesized and evaluated experimentally as our next iteration of inhibitors against Bcr-Abl.

DOI: https://doi.org/10.1021/acs.jpcb.4c02699
Mol Ther. 2024 Jul 01. pii: S1525-0016(24)00386-1. [Epub ahead of print]

Human plasma cells engineered to secrete bispecifics drive effective in vivo leukemia killing.

Tyler F Hill, Parnal Narvekar, Gregory D Asher, Jasmine N Edelstein, Nathan D Camp, Annaiz Grimm, Kerri R Thomas, Michael D Leiken, Katherine M Molloy, Peter J Cook, Sean P Arlauckas, Richard A Morgan, Sarah K Tasian, David J Rawlings, Richard G James.

  Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited in vivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing T cells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1 month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.

Keywords:  T cell engager; bispecific; engineered plasma cells; engineering; engraftment; gene editing; in vivo; leukemia; plasma cells

DOI:  https://doi.org/10.1016/j.ymthe.2024.06.004
bioRxiv. 2024 Jun 18. pii: 2024.06.18.599616. [Epub ahead of print]

The Interplay of Acetylation and Ubiquitination Controls PRMT1 Homeostasis.

Mohd Altaf Najar, Jenna N Beyer, Callie E W Crawford, George M Burslem.

PRMT1 plays many important roles in both normal and disease biology, thus understanding it's regulation is crucial. Herein, we report the role of p300-mediated acetylation at K228 in triggering PRMT1 degradation through FBXL17-mediated ubiquitination. Utilizing mass-spectrometry, cellular biochemistry, and genetic code-expansion technologies, we elucidate a crucial mechanism independent of PRMT1 transcript levels. These results underscore the significance of acetylation in governing protein stability and expand our understanding of PRMT1 homeostasis. By detailing the molecular interplay between acetylation and ubiquitination involved in PRMT1 degradation, this work contributes to broader efforts in deciphering post-translational mechanisms that influence protein homeostasis.

DOI: https://doi.org/10.1101/2024.06.18.599616
bioRxiv. 2024 Jun 18. pii: 2024.06.17.599449. [Epub ahead of print]

Single-nucleus proteomics identifies regulators of protein transport.

Jason Derks, Tobias Jonson, Andrew Leduc, Saad Khan, Luke Khoury, Mahmoud-Reza Rafiee, Nikolai Slavov.

The physiological response of a cell to stimulation depends on its proteome configuration. Therefore, the abundance variation of regulatory proteins across unstimulated single cells can be associatively linked with their response to stimulation. Here we developed an approach that leverages this association across individual cells and nuclei to systematically identify potential regulators of biological processes, followed by targeted validation. Specifically, we applied this approach to identify regulators of nucleocytoplasmic protein transport in macrophages stimulated with lipopolysaccharide (LPS). To this end, we quantified the proteomes of 3,412 individual nuclei, sampling the dynamic response to LPS treatment, and linking functional variability to proteomic variability. Minutes after the stimulation, the protein transport in individual nuclei correlated strongly with the abundance of known protein transport regulators, thus revealing the impact of natural protein variability on functional cellular response. We found that simple biophysical constraints, such as the quantity of nuclear pores, partially explain the variability in LPS-induced nucleocytoplasmic transport. Among the many proteins newly identified to be associated with the response, we selected 16 for targeted validation by knockdown. The knockdown phenotypes confirmed the inferences derived from natural protein and functional variation of single nuclei, thus demonstrating the potential of (sub-)single-cell proteomics to infer functional regulation. We expect this approach to generalize to broad applications and enhance the functional interpretability of single-cell omics data.

DOI: https://doi.org/10.1101/2024.06.17.599449
Methods Mol Biol. 2024 Jul 06.

Identification of De Novo Dividing Stem Cells.

Dmitry I Maltsev, Oleg V Podgorny.

  The ability to alternate between quiescent and proliferating states is a remarkable feature of many types of somatic stem cells. The balance between quiescent and proliferating states is vital for maintenance of stem cells over the lifespan, and its disturbance may lead to premature depletion of the stem cell pool and loss of the tissue regenerative or renewal capacity at later stages of life. The question on how this balance is regulated is of critical importance in stem cell research and biology of aging. Assessment of the balance between quiescent and proliferating states has remained challenged until recently due to the lack of approaches for robust determination of the rate at which stem cells exit reversible cell cycle arrest. Here, we propose a simple method for detection of those stem cells that have entered the division cycle after a prolonged period of quiescence.The method combines cumulative and pulse labeling with thymidine analogues 5-bromo-2'-deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU). In the discussed labeling scheme, cells that have incorporated only the second label, EdU, are de novo dividing cells. The suggested double labeling method provides quantitative assessment of the rate at which stem cells exit the quiescent state and allows the fates of de novo dividing stem cells to be traced.

Keywords:  Aging; Cell cycle; Osmotic minipumps; Quiescent stem cells; Thymidine analogues

DOI:  https://doi.org/10.1007/7651_2024_560
J Gene Med. 2024 Jul;26(7): e3717

Hematopoietic stem cell gene therapy for the treatment of SYNGAP1-related non-specific intellectual disability.

Joseph S Anderson, Alyse L Lodigiani, Camilla M Barbaduomo, Julie R Beegle.

   BACKGROUND: Synaptic Ras GTPase activating protein 1 (SYNGAP1)-related non-specific intellectual disability is a neurodevelopmental disorder caused by an insufficient level of SynGAP1 resulting in a dysfunction of neuronal synapses and presenting with a wide array of clinical phenotypes. Hematopoietic stem cell gene therapy has the potential to deliver therapeutic levels of functional SynGAP1 to affected neurons upon transduction of hematopoietic stem and progenitor cells with a lentiviral vector.
METHODS: As a novel approach toward the treatment of SYNGAP1, we have generated a lentiviral vector expressing a modified form of SynGAP1 for transduction of human CD34+ hematopoietic stem and progenitor cells. The gene-modified cells were then transplanted into adult immunodeficient SYNGAP1+/- heterozygous mice and evaluated for improvement of SYNGAP1-related clinical phenotypes. Expression of SynGAP1 was also evaluated in the brain tissue of transplanted mice.
RESULTS: In our proof-of-concept study, we have demonstrated significant improvement of SYNGAP1-related phenotypes including an improvement in motor abilities observed in mice transplanted with the vector transduced cells because they displayed decreased hyperactivity in an open field assay and an increased latency to fall in a rotarod assay. An increased level of SynGAP1 was also detected in the brains of these mice.
CONCLUSIONS: These early-stage results highlight the potential of this stem cell gene therapy approach as a treatment strategy for SYNGAP1.

Keywords:  SYNGAP1; gene therapy; hematopoietic stem cells; lentiviral vector; neurodevelopmental disorders

DOI:  https://doi.org/10.1002/jgm.3717
Trends Biochem Sci. 2024 Jun 29. pii: S0968-0004(24)00149-X. [Epub ahead of print]

UPS-dependent strategies of protein quality control degradation.

Leonie Müller, Thorsten Hoppe.

  The degradation of damaged proteins is critical for tissue integrity and organismal health because damaged proteins have a high propensity to form aggregates. E3 ubiquitin ligases are key regulators of protein quality control (PQC) and mediate the selective degradation of damaged proteins, a process termed 'PQC degradation' (PQCD). The degradation signals (degrons) that trigger PQCD are based on hydrophobic sites that are normally buried within the native protein structure. However, an open question is how PQCD-specialized E3 ligases distinguish between transiently misfolded proteins, which can be efficiently refolded, and permanently damaged proteins, which must be degraded. While significant progress has been made in characterizing degradation determinants, understanding the key regulatory signals of cellular and organismal PQCD pathways remains a challenge.

Keywords:  E3 ubiquitin ligase; degron; molecular chaperone; protein damage; proteostasis; ubiquitin–proteasome system (UPS)

DOI:  https://doi.org/10.1016/j.tibs.2024.06.006
Genes Dis. 2024 Sep;11(5): 101150

Exploiting the potential of the ubiquitin-proteasome system in overcoming tyrosine kinase inhibitor resistance in chronic myeloid leukemia.

Xudong Li, Wei Li, Yanli Zhang, Linping Xu, Yongping Song.

  The advent of tyrosine kinase inhibitors (TKI) targeting BCR-ABL has drastically changed the treatment approach of chronic myeloid leukemia (CML), greatly prolonged the life of CML patients, and improved their prognosis. However, TKI resistance is still a major problem with CML patients, reducing the efficacy of treatment and their quality of life. TKI resistance is mainly divided into BCR-ABL-dependent and BCR-ABL-independent resistance. Now, the main clinical strategy addressing TKI resistance is to switch to newly developed TKIs. However, data have shown that these new drugs may cause serious adverse reactions and intolerance and cannot address all resistance mutations. Therefore, finding new therapeutic targets to overcome TKI resistance is crucial and the ubiquitin-proteasome system (UPS) has emerged as a focus. The UPS mediates the degradation of most proteins in organisms and controls a wide range of physiological processes. In recent years, the study of UPS in hematological malignant tumors has resulted in effective treatments, such as bortezomib in the treatment of multiple myeloma and mantle cell lymphoma. In CML, the components of UPS cooperate or antagonize the efficacy of TKI by directly or indirectly affecting the ubiquitination of BCR-ABL, interfering with CML-related signaling pathways, and negatively or positively affecting leukemia stem cells. Some of these molecules may help overcome TKI resistance and treat CML. In this review, the mechanism of TKI resistance is briefly described, the components of UPS are introduced, existing studies on UPS participating in TKI resistance are listed, and UPS as the therapeutic target and strategies are discussed.

Keywords:  Chronic myeloid leukemia; Deubiquitinases; E3 ligase; PROTAC; TKI resistance; Ubiquitin-proteasome system

DOI:  https://doi.org/10.1016/j.gendis.2023.101150
Nat Mater. 2024 Jul 04.

Engineered matrices reveal stiffness-mediated chemoresistance in patient-derived pancreatic cancer organoids.

Bauer L LeSavage, Daiyao Zhang, Carla Huerta-López, Aidan E Gilchrist, Brad A Krajina, Kasper Karlsson, Amber R Smith, Kremena Karagyozova, Katarina C Klett, Michelle S Huang, Christopher Long, Gernot Kaber, Christopher M Madl, Paul L Bollyky, Christina Curtis, Calvin J Kuo, Sarah C Heilshorn.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its fibrotic and stiff extracellular matrix. However, how the altered cell/extracellular-matrix signalling contributes to the PDAC tumour phenotype has been difficult to dissect. Here we design and engineer matrices that recapitulate the key hallmarks of the PDAC tumour extracellular matrix to address this knowledge gap. We show that patient-derived PDAC organoids from three patients develop resistance to several clinically relevant chemotherapies when cultured within high-stiffness matrices mechanically matched to in vivo tumours. Using genetic barcoding, we find that while matrix-specific clonal selection occurs, cellular heterogeneity is not the main driver of chemoresistance. Instead, matrix-induced chemoresistance occurs within a stiff environment due to the increased expression of drug efflux transporters mediated by CD44 receptor interactions with hyaluronan. Moreover, PDAC chemoresistance is reversible following transfer from high- to low-stiffness matrices, suggesting that targeting the fibrotic extracellular matrix may sensitize chemoresistant tumours. Overall, our findings support the potential of engineered matrices and patient-derived organoids for elucidating extracellular matrix contributions to human disease pathophysiology.

DOI: https://doi.org/10.1038/s41563-024-01908-x
Elife. 2024 Jul 04. pii: e84798. [Epub ahead of print]13

Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum.

Ya-Juan Wang, Xiao-Jing Di, Pei-Pei Zhang, Xi Chen, Marnie P Williams, Dong-Yun Han, Raad Nashmi, Brandon J Henderson, Fraser J Moss, Ting-Wei Mu.

  Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.

Keywords:  biochemistry; cell biology; chemical biology; human; mouse

DOI:  https://doi.org/10.7554/eLife.84798
Cancer Innov. 2024 Jun;3(3): e118

Establishment of a humanized mouse model using steady-state peripheral blood-derived hematopoietic stem and progenitor cells facilitates screening of cancer-targeted T-cell repertoires.

Yulin Xu, Wei Shan, Qian Luo, Meng Zhang, Dawei Huo, Yijin Chen, Honghu Li, Yishan Ye, Xiaohong Yu, Yi Luo, He Huang.

   Background: Cancer-targeted T-cell receptor T (TCR-T) cells hold promise in treating cancers such as hematological malignancies and breast cancers. However, approaches to obtain cancer-reactive TCR-T cells have been unsuccessful.
Methods: Here, we developed a novel strategy to screen for cancer-targeted TCR-T cells using a special humanized mouse model with person-specific immune fingerprints. Rare steady-state circulating hematopoietic stem and progenitor cells were expanded via three-dimensional culture of steady-state peripheral blood mononuclear cells, and then the expanded cells were applied to establish humanized mice. The human immune system was evaluated according to the kinetics of dendritic cells, monocytes, T-cell subsets, and cytokines. To fully stimulate the immune response and to obtain B-cell precursor NAML-6- and triple-negative breast cancer MDA-MB-231-targeted TCR-T cells, we used the inactivated cells above to treat humanized mice twice a day every 7 days. Then, human T cells were processed for TCR β-chain (TRB) sequencing analysis. After the repertoires had been constructed, features such as the fraction, diversity, and immune signature were investigated.
Results: The results demonstrated an increase in diversity and clonality of T cells after treatment. The preferential usage and features of TRBV, TRBJ, and the V-J combination were also changed. The stress also induced highly clonal expansion. Tumor burden and survival analysis demonstrated that stress induction could significantly inhibit the growth of subsequently transfused live tumor cells and prolong the survival of the humanized mice.
Conclusions: We constructed a personalized humanized mouse model to screen cancer-targeted TCR-T pools. Our platform provides an effective source of cancer-targeted TCR-T cells and allows for the design of patient-specific engineered T cells. It therefore has the potential to greatly benefit cancer treatment.

Keywords:  T‐cell receptor β‐chain (TRB); cancer‐targeted T‐cell receptor T (TCR‐T) cells; circulating hematopoietic stem and progenitor cells (HSPCs); humanized mouse model; steady‐state peripheral blood; three‐dimensional culture

DOI:  https://doi.org/10.1002/cai2.118
Redox Biol. 2024 Jun 27. pii: S2213-2317(24)00237-4. [Epub ahead of print]75 103259

Protein modification and degradation in ferroptosis.

Yuan Wang, Ding Yan, Jinbao Liu, Daolin Tang, Xin Chen.

  Ferroptosis is a form of iron-related oxidative cell death governed by an integrated redox system, encompassing pro-oxidative proteins and antioxidative proteins. These proteins undergo precise control through diverse post-translational modifications, including ubiquitination, phosphorylation, acetylation, O-GlcNAcylation, SUMOylation, methylation, N-myristoylation, palmitoylation, and oxidative modification. These modifications play pivotal roles in regulating protein stability, activity, localization, and interactions, ultimately influencing both the buildup of iron and lipid peroxidation. In mammalian cells, regulators of ferroptosis typically undergo degradation via two principal pathways: the ubiquitin-proteasome system, which handles the majority of protein degradation, and autophagy, primarily targeting long-lived or aggregated proteins. This comprehensive review aims to summarize recent advances in the post-translational modification and degradation of proteins linked to ferroptosis. It also discusses strategies for modulating ferroptosis through protein modification and degradation systems, providing new insights into potential therapeutic applications for both cancer and non-neoplastic diseases.

Keywords:  Autophagy; Degradation; Ferroptosis; Modification; Proteasome

DOI:  https://doi.org/10.1016/j.redox.2024.103259
Eur J Haematol. 2024 Jul 05.

Aberrant early hematopoietic progenitor formation marks the onset of hematopoietic defects in Shwachman-Diamond syndrome.

Alejandra Lagos-Monzon, Stephanie Ng, Alice M Luca, Hongbing Li, Mathura Sabanayagam, Mariana Benicio, Houtan Moshiri, Richard Armstrong, Chetan Tailor, Marion Kennedy, Eyal Grunebaum, Gordon Keller, Yigal Dror.

  Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure disorder that often presents at infancy. Progress has been made in revealing causal mutated genes (SBDS and others), ribosome defects, and hematopoietic aberrations in SDS. However, the mechanism underlying the hematopoietic failure remained unknown, and treatment options are limited. Herein, we investigated the onset of SDS embryonic hematopoietic impairments. We generated SDS and control human-derived induced pluripotent stem cells (iPSCs). SDS iPSCs recapitulated the SDS hematological phenotype. Detailed stepwise evaluation of definitive hematopoiesis revealed defects that started at the early emerging hematopoietic progenitor (EHP) stage after mesoderm and hemogenic endothelium were normally induced. Hematopoietic potential of EHPs was markedly reduced, and the introduction of SBDS in SDS iPSCs improved colony formation. Transcriptome analysis revealed reduced expression of ribosome and oxidative phosphorylation-related genes in undifferentiated and differentiated iPSCs. However, certain pathways (e.g., DNA replication) and genes (e.g., CHCHD2) were exclusively or more severely dysregulated in EHPs compared with earlier and later stages. To our knowledge, this study offers for the first time an insight into the embryonic onset of human hematopoietic defects in an inherited bone marrow failure syndrome and reveals cellular and molecular aberrations at critical stages of hematopoietic development toward EHPs.

Keywords:  Shwachman–Diamond syndrome; bone marrow failure; differentiation; disease modelling; hematopoietic stem cell; human induced pluripotent stem cells; transcriptional analysis

DOI:  https://doi.org/10.1111/ejh.14260