bims-scepro 2024-07-21 papers

bims-scepro

Biomed News

on Stem cell proteostasis

Issue of 2024‒07‒21
35 papers selected by
William Grey, University of York

Mobilization dynamics of bone marrow hematopoietic stem cells during hematopoietic regeneration.
Nudt15-mediated inflammatory signaling contributes to divergent outcomes in leukemogenesis and hematopoiesis.
The molecular and cellular hematopoietic stem cell specification niche.
Integrin-α9 overexpression underlies the niche-independent maintenance of leukemia stem cells in acute myeloid leukemia.
Terminal deoxynucleotidyl transferase and CD84 identify human multi-potent lymphoid progenitors.
A metabolic atlas of blood cells in young and aged mice identifies uridine as a metabolite to rejuvenate aged hematopoietic stem cells.
PHF6 suppresses self-renewal of leukemic stem cells in AML.
Counting blood precursors.
RHOF activation of AKT/β-catenin signaling pathway drives acute myeloid leukemia progression and chemotherapy resistance.
Umbilical Cord Blood Hematopoietic Cells: From Biology to Hematopoietic Transplants and Cellular Therapies.
Characterization of immortalized bone marrow erythroid progenitor adult (imBMEP-A)-The first inducible immortalized red blood cell progenitor cell line derived from bone marrow CD71-positive cells.
Stochastic journeys of cell progenies through compartments and the role of self-renewal, symmetric and asymmetric division.
Targeting the Sodium-Potassium Pump as a Therapeutic Strategy in Acute Myeloid Leukemia.
Proteomic analysis reveals a PLK1-dependent G2/M degradation program and a role for AKAP2 in coordinating the mitotic cytoskeleton.
Diversity of post-translational modifications and cell signaling revealed by single cell and single organelle mass spectrometry.
Proteomic screening identifies PF4/Cxcl4 as a critical driver of myelofibrosis.
CryoET reveals actin filaments within platelet microtubules.
MYB: A Key Transcription Factor in the Hematopoietic System Subject to Many Levels of Control.
DropBlot: single-cell western blotting of chemically fixed cancer cells.
Frontline therapy of acute myeloid leukemia with lower intensity regimens: Where are we now and where can we go?
Assessing the Survival of Human Plasma Cells Ex Vivo.
Interference with PLA2G16 promotes cell cycle arrest and apoptosis and inhibits the reprogramming of glucose metabolism in multiple myeloma cells by modulating the Hippo/YAP signaling pathway.
MLL-AF9 regulates transcriptional initiation in mixed lineage leukemic cells.
circPCMTD1 : A protein-coding circular RNA that regulates DNA damage response in BCR/ABL -positive leukemias.
The erlin1/erlin2 complex binds to and stabilizes phosphatidylinositol 3-phosphate and regulates autophagy.
Pathways to therapy resistance: The sheltering effect of the bone marrow microenvironment to multiple myeloma cells.
FLT3/CD99 Bispecific antibody-based nanoparticles (BiAbs) for Acute Myeloid Leukemia.
Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation.
Centrosome age breaks spindle size symmetry even in cells thought to divide symmetrically.
Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms.
Genomic and immune determinants of resistance to daratumumab-based therapy in relapsed refractory multiple myeloma.
Clonal inactivation of TERT impairs stem cell competition.
Benchmarking and Automating the Biotinylation Proteomics Workflow.
Long-Term Culture of Human Pluripotent Stem Cells in Xeno-Free Condition Using Functional Polymer Films.
A Hypochlorite-Activated Theranostic Prodrug for Selective Imaging of High Myeloperoxidase Expression Acute Myeloid Leukemia Cells and Drug Release.

Exp Hematol. 2024 Jul 13. pii: S0301-472X(24)00140-1. [Epub ahead of print] 104281

Mobilization dynamics of bone marrow hematopoietic stem cells during hematopoietic regeneration.

Alban Johansson, Ahad Khalilnezhad, Hitoshi Takizawa, Hidenobu Mizuno, Toshio Suda, Terumasa Umemoto.

  Under stress hematopoiesis, previous studies have suggested the migration of hematopoietic stem cells (HSCs) from bone marrow (BM) to extramedullary sites such as spleen. However, there is little direct evidence of HSC migration from BM to spleen. Here, we induced myeloablation via 5-fluorouracil (5-FU) and showed the direct evidence of HSC migration from BM to spleen during hematopoietic regeneration via a photoconvertible fluorophore. Moreover, during steady-state, HSCs preferentially migrated to BM rather than spleen, but during hematopoietic regeneration HSCs preferred to spleen as a migration site equivalently or greater. Furthermore, in the early phase, HSCs egressed from BM through the attenuated HSC retention. However, HSCs in the late phase gained significantly enhanced cell-autonomous motility, which was independent of chemotaxis. Collectively, HSC mobilization from BM prior to the migration to spleen was dynamically changed from passive to active events during hematopoietic regeneration.

Keywords:  Bone marrow regeneration; Cell migration; Extramedullary hematopoiesis; Hematopoietic stem cell

DOI:  https://doi.org/10.1016/j.exphem.2024.104281
Leukemia. 2024 Jul 18.

Nudt15-mediated inflammatory signaling contributes to divergent outcomes in leukemogenesis and hematopoiesis.

Jiachen Wang, Yu Zhang, Lei Li, Liujiao Wang, Shuainan Sun, Bowu Wang, Yanwen Ge, Zhonghui Zhang.

NUDT15 encodes nucleotide triphosphate diphosphatase that is responsible for metabolizing purine analog drugs, and its genetic mutation results in severe side effects from thiopurine therapy. However, the functions of Nudt15 in leukemic stem cells (LSCs) and hematopoietic stem cells (HSCs) remain unknown. Here we reveal the Nudt15-regulating self-renewal of both mouse LSCs and HSCs. Our data show that Nudt15 negatively regulates murine leukemogenesis and its deficiency prolongs the survival of murine AML recipients by impairing LSC self-renewal, while Nudt15 ablation markedly enhances mouse HSC regenerative potential and self-renewal. Mechanistically, Nudt15 modulates inflammatory signaling in mouse LSCs and HSCs, leading to divergent self-renewal outcomes. Nudt15 depletion inhibits mouse LSC self-renewal by downregulating Ifi30, resulting in elevating intracellular ROS level. Gata2, a key regulator, is required for Nudt15-mediating inflammatory signaling in mouse HSCs. Collectively, our results present new crucial roles of Nudt15 in maintaining the functions of mouse LSC and HSC through inflammatory signaling and have a new insight into clinical implications.

DOI: https://doi.org/10.1038/s41375-024-02352-1
Exp Hematol. 2024 Jul 13. pii: S0301-472X(24)00139-5. [Epub ahead of print] 104280

The molecular and cellular hematopoietic stem cell specification niche.

Wilson K Clements, Hanane Khoury.

  Hematopoietic stem cells (HSCs) are a population of tissue-specific stem cells that reside in the bone marrow of adult mammals where they self-renew and continuously regenerate the adult hematopoietic lineages over the life of the individual. Prominence as a stem cell model and clinical usefulness has driven interest in understanding the physiological processes that lead to specification of HSCs during embryonic development. High efficiency directed differentiation of HSCs by instruction of defined progenitor cells using sequentially defined instructive molecules and conditions remains impossible, indicating that comprehensive knowledge of the complete set of precursor intermediate identities and required inductive inputs remains incompletely understood. Recently, interest in the molecular and cellular microenvironment where HSCs are specified from endothelial precursors-the "specification niche"-has increased. Here we review recent progress in understanding these niche spaces across vertebrate phyla, as well as how a better characterization of the origin and molecular phenotypes of the niche cell populations has helped inform and complicate previous understanding of signaling required for HSC emergence and maturation.

Keywords:  AGM; BMP; CHT; FGF; HSC; Mouse; Notch; VSMC; WNT; aorta gonads mesonephros; caudal hematopoietic tissue; developmental hematopoiesis; dorsal aorta; fetal liver; hematopoietic stem cell; inflammatory signaling; mesoderm; microenvironment; neural crest; sclerotome; somite; specification; splanchnopleural mesoderm; vascular smooth muscle cells; vasculature; zebrafish chick

DOI:  https://doi.org/10.1016/j.exphem.2024.104280
Gene. 2024 Jul 11. pii: S0378-1119(24)00642-5. [Epub ahead of print]928 148761

Integrin-α9 overexpression underlies the niche-independent maintenance of leukemia stem cells in acute myeloid leukemia.

Akiko Niibori-Nambu, Chelsia Qiuxia Wang, Desmond Wai Loon Chin, Jing Yuan Chooi, Hiroki Hosoi, Takashi Sonoki, Cheng-Yong Tham, Giselle Sek Suan Nah, Branko Cirovic, Darren Qiancheng Tan, Hitoshi Takizawa, Goro Sashida, Yufen Goh, Jiaqi Tng, Wee Nih Fam, Melissa Jane Fullwood, Toshio Suda, Henry Yang, Vinay Tergaonkar, Ichiro Taniuchi, Shang Li, Wee Joo Chng, Motomi Osato.

  Leukemia stem cells (LSCs) are widely believed to reside in well-characterized bone marrow (BM) niches; however, the capacity of the BM niches to accommodate LSCs is insufficient, and a significant proportion of LSCs are instead maintained in regions outside the BM. The molecular basis for this niche-independent behavior of LSCs remains elusive. Here, we show that integrin-α9 overexpression (ITGA9 OE) plays a pivotal role in the extramedullary maintenance of LSCs by molecularly mimicking the niche-interacting status, through the binding with its soluble ligand, osteopontin (OPN). Retroviral insertional mutagenesis conducted on leukemia-prone Runx-deficient mice identified Itga9 OE as a novel leukemogenic event. Itga9 OE activates Akt and p38MAPK signaling pathways. The elevated Myc expression subsequently enhances ribosomal biogenesis to overcome the cell integrity defect caused by the preexisting Runx alteration. The Itga9-Myc axis, originally discovered in mice, was further confirmed in multiple human acute myeloid leukemia (AML) subtypes, other than RUNX leukemias. In addition, ITGA9 was shown to be a functional LSC marker of the best prognostic value among 14 known LSC markers tested. Notably, the binding of ITGA9 with soluble OPN, a known negative regulator against HSC activation, induced LSC dormancy, while the disruption of ITGA9-soluble OPN interaction caused rapid cell propagation. These findings suggest that the ITGA9 OE increases both actively proliferating leukemia cells and dormant LSCs in a well-balanced manner, thereby maintaining LSCs. The ITGA9 OE would serve as a novel therapeutic target in AML.

Keywords:  ITGA9; Leukemia stem cell; Osteopontin; Runx

DOI:  https://doi.org/10.1016/j.gene.2024.148761
Nat Commun. 2024 Jul 13. 15(1): 5910

Terminal deoxynucleotidyl transferase and CD84 identify human multi-potent lymphoid progenitors.

YeEun Kim, Ariel A Calderon, Patricia Favaro, David R Glass, Albert G Tsai, Daniel Ho, Luciene Borges, William J Greenleaf, Sean C Bendall.

Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features, we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase intrinsic to VDJ recombination, broadly expressed within CD34+ progenitors prior to B/T cell emergence. While these TdT+ cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype, their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84lo GMPs demonstrates robust lymphoid potentials ex vivo, while still retaining significant myeloid differentiation capacity, akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors, further defining the lympho-myeloid axis in human hematopoiesis.

DOI: https://doi.org/10.1038/s41467-024-49883-w
Nat Aging. 2024 Jul 17.

A metabolic atlas of blood cells in young and aged mice identifies uridine as a metabolite to rejuvenate aged hematopoietic stem cells.

Xiangjun Zeng, Ce Shi, Yingli Han, Kejia Hu, Xiaoqing Li, Cong Wei, Lijuan Ding, Jiazhen Cui, Simao Huang, Yulin Xu, Meng Zhang, Wei Shan, Qian Luo, Jian Yu, Zhongzheng Zheng, Xia Li, Pengxu Qian, He Huang.

Aging of hematopoietic stem cells (HSCs) is accompanied by impaired self-renewal ability, myeloid skewing, immunodeficiencies and increased susceptibility to malignancies. Although previous studies highlighted the pivotal roles of individual metabolites in hematopoiesis, comprehensive and high-resolution metabolomic profiles of different hematopoietic cells across ages are still lacking. In this study, we created a metabolome atlas of different blood cells across ages in mice. We reveal here that purine, pyrimidine and retinol metabolism are enriched in young hematopoietic stem and progenitor cells (HSPCs), whereas glutamate and sphingolipid metabolism are concentrated in aged HSPCs. Through metabolic screening, we identified uridine as a potential regulator to rejuvenate aged HSPCs. Mechanistically, uridine treatment upregulates the FoxO signaling pathway and enhances self-renewal while suppressing inflammation in aged HSCs. Finally, we constructed an open-source platform for public easy access and metabolomic analysis in blood cells. Collectively, we provide a resource for metabolic studies in hematopoiesis that can contribute to future anti-aging metabolite screening.

DOI: https://doi.org/10.1038/s43587-024-00669-1
Leukemia. 2024 Jul 14.

PHF6 suppresses self-renewal of leukemic stem cells in AML.

Sapana S Jalnapurkar, Aishwarya S Pawar, Subin S George, Charles Antony, Patrick Somers, Jason Grana, Victoria K Feist, Sandeep Gurbuxani, Vikram R Paralkar.

Acute myeloid leukemia is characterized by uncontrolled proliferation of self-renewing myeloid progenitors accompanied by a differentiation arrest. PHF6 is a chromatin-binding protein mutated in myeloid leukemias, and its isolated loss increases mouse HSC self-renewal without malignant transformation. We report here that Phf6 knockout increases the aggressiveness of Hoxa9-driven AML over serial transplantation, and increases the frequency of leukemia initiating cells. We define the in vivo hierarchy of Hoxa9-driven AML and identify a population that we term the "LIC-e" (leukemia initiating cells enriched) population. We find that Phf6 loss expands the LIC-e population and skews its transcriptome to a more stem-like state; concordant transcriptome shifts are also observed on PHF6 knockout in a human AML cell line and in PHF6 mutant patient samples from the BEAT AML dataset. We demonstrate that LIC-e accumulation in Phf6 knockout AML occurs not due to effects on cell cycle or apoptosis, but due to an increase in the fraction of its progeny that retain LIC-e identity. Our work indicates that Phf6 loss increases AML self-renewal through context-specific effects on leukemia stem cells.

DOI: https://doi.org/10.1038/s41375-024-02340-5
Elife. 2024 Jul 17. pii: e100373. [Epub ahead of print]13

Counting blood precursors.

Sarah Duchamp de Chastaigne, Catherine M Sawai.

  A new mathematical model can estimate the number of precursor cells that contribute to regenerating blood cells in mice.

Keywords:  HSPCs; blood; bone marrow; hematopoiesis; hematopoietic stem; mouse; progenitor cells; regenerative medicine; stem cells

DOI:  https://doi.org/10.7554/eLife.100373
iScience. 2024 Jul 19. 27(7): 110221

RHOF activation of AKT/β-catenin signaling pathway drives acute myeloid leukemia progression and chemotherapy resistance.

Xin Wen, Peng Li, Yuechan Ma, Dongmei Wang, Ruinan Jia, Yuan Xia, Wei Li, Yongjian Li, Guosheng Li, Tao Sun, Fei Lu, Jingjing Ye, Chunyan Ji.

  Acute myeloid leukemia (AML) is a clonal malignancy originating from leukemia stem cells, characterized by a poor prognosis, underscoring the necessity for novel therapeutic targets and treatment methodologies. This study focuses on Ras homolog family member F, filopodia associated (RHOF), a Rho guanosine triphosphatase (GTPase) family member. We found that RHOF is overexpressed in AML, correlating with an adverse prognosis. Our gain- and loss-of-function experiments revealed that RHOF overexpression enhances proliferation and impedes apoptosis in AML cells in vitro. Conversely, genetic suppression of RHOF markedly reduced the leukemia burden in a human AML xenograft mouse model. Furthermore, we investigated the synergistic effect of RHOF downregulation and chemotherapy, demonstrating significant therapeutic efficacy in vivo. Mechanistically, RHOF activates the AKT/β-catenin signaling pathway, thereby accelerating the progression of AML. Our findings elucidate the pivotal role of RHOF in AML pathogenesis and propose RHOF inhibition as a promising therapeutic approach for AML management.

Keywords:  Biochemistry; Cancer; Cell biology; Molecular biology

DOI:  https://doi.org/10.1016/j.isci.2024.110221
Arch Med Res. 2024 Jul 13. pii: S0188-4409(24)00094-8. [Epub ahead of print]55(6): 103042

Umbilical Cord Blood Hematopoietic Cells: From Biology to Hematopoietic Transplants and Cellular Therapies.

Hector Mayani.

  Umbilical cord blood (UCB) is a rich source of hematopoietic stem and progenitor cells that are biologically superior to their adult counterparts. UCB cells can be stored for several years without compromising their numbers or function. Today, public and private UCB banks have been established in several countries around the world. After 35 years since the first UCB transplant (UCBT), more than 50,000 UCBTs have been performed worldwide. In pediatric patients, UCBT is comparable to or superior to bone marrow transplantation. In adult patients, UCB can be an alternative source of hematopoietic cells when an HLA-matched unrelated adult donor is not available and when a transplant is urgently needed. Delayed engraftment (due to reduced absolute numbers of hematopoietic cells) and higher costs have led many medical institutions not to consider UCB as a first-line cell source for hematopoietic transplants. As a result, the use of UCB as a source of hematopoietic stem and progenitor cells for transplantation has declined over the past decade. Several approaches are being investigated to make UCBTs more efficient, including improving the homing capabilities of primitive UCB cells and increasing the number of hematopoietic cells to be infused. Several of these approaches have already been applied in the clinic with promising results. UCB also contains immune effector cells, including monocytes and various lymphocyte subsets, which, together with stem and progenitor cells, are excellent candidates for the development of cellular therapies for hematological and non-hematological diseases.

Keywords:  Cellular therapies; Cord blood banking; Cord blood cells; Hematopoietic stem/progenitor cells; Hematopoietic transplants; Immune cells

DOI:  https://doi.org/10.1016/j.arcmed.2024.103042
Cytotherapy. 2024 Jul 02. pii: S1465-3249(24)00767-9. [Epub ahead of print]

Characterization of immortalized bone marrow erythroid progenitor adult (imBMEP-A)-The first inducible immortalized red blood cell progenitor cell line derived from bone marrow CD71-positive cells.

Romy Kronstein-Wiedemann, Jessica Thiel, Duran Sürün, Madeleine Teichert, Stephan R Künzel, Stefan Zimmermann, Lisa Wagenführ, Frank Buchholz, Torsten Tonn.

  BACKGROUND AIMS: Ex vivo production of red blood cells (RBCs) represents a promising alternative for transfusion medicine. Several strategies have been described to generate erythroid cell lines from different sources, including embryonic, induced pluripotent, and hematopoietic stem cells. All these approaches have in common that they require elaborate differentiation cultures whereas the yield of enucleated RBCs is inefficient.METHODS: We generated a human immortalized adult erythroid progenitor cell line derived from bone marrow CD71-positive erythroid progenitor cells (immortalized bone marrow erythroid progenitor adult, or imBMEP-A) by an inducible expression system, to shorten differentiation culture necessary for terminal erythroid differentiation. It is the first erythroid cell line that is generated from direct reticulocyte progenitors and demonstrates robust hemoglobin production in the immortalized state.
RESULTS: Morphologic analysis of the immortalized cells showed that the preferred cell type of the imBMEP-A line corresponds to hemoglobin-producing basophilic erythroblasts. In addition, we were able to generate a stable cell line from a single cell clone with the triple knockout of RhAG, RhDCE and KELL. After removal of doxycycline, part of the cells differentiated into normoblasts and reticulocytes within 5-7 days.
CONCLUSIONS: Our results demonstrate that the imBMEP-A cell line can serve as a stable and straightforward modifiable platform for RBC engineering in the future.

Keywords:  Blood pharming; Cell line; Erythrocyte; Transfusion; Universal blood

DOI:  https://doi.org/10.1016/j.jcyt.2024.06.009
Sci Rep. 2024 Jul 15. 14(1): 16287

Stochastic journeys of cell progenies through compartments and the role of self-renewal, symmetric and asymmetric division.

Hanan Dreiwi, Flavia Feliciangeli, Mario Castro, Grant Lythe, Carmen Molina-París, Martín López-García.

Division and differentiation events by which cell populations with specific functions are generated often take place as part of a developmental programme, which can be represented by a sequence of compartments. A compartment is the set of cells with common characteristics; sharing, for instance, a spatial location or a phenotype. Differentiation events are transitions from one compartment to the next. Cells may also die or divide. We consider three different types of division events: (i) where both daughter cells inherit the mother's phenotype (self-renewal), (ii) where only one of the daughters changes phenotype (asymmetric division), and (iii) where both daughters change phenotype (symmetric division). The self-renewal probability in each compartment determines whether the progeny of a single cell, moving through the sequence of compartments, is finite or grows without bound. We analyse the progeny stochastic dynamics with probability generating functions. In the case of self-renewal, by following one of the daughters after any division event, we may construct lifelines containing only one cell at any time. We analyse the number of divisions along such lines, and the compartment where lines terminate with a death event. Analysis and numerical simulations are applied to a five-compartment model of the gradual differentiation of hematopoietic stem cells and to a model of thymocyte development: from pre-double positive to single positive (SP) cells with a bifurcation to either SP4 or SP8 in the last compartment of the sequence.

DOI: https://doi.org/10.1038/s41598-024-63500-2
Cancer Res. 2024 Jul 18.

Targeting the Sodium-Potassium Pump as a Therapeutic Strategy in Acute Myeloid Leukemia.

Constanze Schneider, Hermes Spaink, Gabriela Alexe, Neekesh V Dharia, Ashleigh Meyer, Lucy A Merickel, Delan Khalid, Sebastian Scheich, Bjorn Haupl, Louis M Staudt, Thomas Oellerich, Kimberly Stegmaier.

Tissue-specific differences in the expression of paralog genes, which are not essential in most cell types due to the buffering effect of the partner pair, can make for highly selective gene dependencies. To identify selective paralogous targets for acute myeloid leukemia (AML), we integrated the Cancer Dependency Map with numerous datasets characterizing protein-protein interactions, paralog relationships, and gene expression in cancer models. Here, we identified ATP1B3 as a context-specific, paralog-related dependency in AML. ATP1B3, the beta subunit of the sodium-potassium pump (Na/K-ATP pump), interacts with the alpha subunit ATP1A1 to form an essential complex for maintaining cellular homeostasis and membrane potential in all eukaryotic cells. When ATP1B3's paralog ATP1B1 is poorly expressed, elimination of ATP1B3 leads to the destabilization of the Na/K-ATP pump. ATP1B1 expression is regulated through epigenetic silencing in hematopoietic lineage cells both through histone and DNA methylation in the promoter region. Loss of ATP1B3 in AML cells induced cell death in vitro and reduced leukemia burden in vivo, which could be rescued by stabilizing ATP1A1 through overexpression of ATP1B1. ATP1B3 is thus a potential therapeutic target for AML and other hematologic malignancies with low expression of ATP1B1.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-3560
Cell Rep. 2024 Jul 16. pii: S2211-1247(24)00839-8. [Epub ahead of print]43(8): 114510

Proteomic analysis reveals a PLK1-dependent G2/M degradation program and a role for AKAP2 in coordinating the mitotic cytoskeleton.

Ryan D Mouery, Kimberly Lukasik, Carolyn Hsu, Thomas Bonacci, Derek L Bolhuis, Xianxi Wang, C Allie Mills, E Drew Toomer, Owen G Canterbury, Kevin C Robertson, Timothy B Branigan, Nicholas G Brown, Laura E Herring, Stephanie L Gupton, Michael J Emanuele.

  Ubiquitination is an essential regulator of cell division. The kinase Polo-like kinase 1 (PLK1) promotes protein degradation at G2/M phase through the E3 ubiquitin ligase Skp1-Cul1-F box (SCF)βTrCP. However, the magnitude to which PLK1 shapes the mitotic proteome is uncharacterized. Combining quantitative proteomics with pharmacologic PLK1 inhibition revealed a widespread, PLK1-dependent program of protein breakdown at G2/M. We validated many PLK1-regulated proteins, including substrates of the cell-cycle E3 SCFCyclin F, demonstrating that PLK1 promotes proteolysis through at least two distinct E3 ligases. We show that the protein-kinase-A-anchoring protein A-kinase anchor protein 2 (AKAP2) is cell-cycle regulated and that its mitotic degradation is dependent on the PLK1/βTrCP signaling axis. Expression of a non-degradable AKAP2 mutant resulted in actin defects and aberrant mitotic spindles, suggesting that AKAP2 degradation coordinates cytoskeletal organization during mitosis. These findings uncover PLK1's far-reaching role in shaping the mitotic proteome post-translationally and have potential implications in malignancies where PLK1 is upregulated.

Keywords:  AKAP2; CP: Cell biology; CP: Molecular biology; Cullin RING Ligase; Cyclin F; G2/M; Plk1; cell cycle; mitosis; protein degradation; ubiquitin; βTRCP

DOI:  https://doi.org/10.1016/j.celrep.2024.114510
Commun Biol. 2024 Jul 19. 7(1): 884

Diversity of post-translational modifications and cell signaling revealed by single cell and single organelle mass spectrometry.

Dong-Gi Mun, Firdous A Bhat, Neha Joshi, Leticia Sandoval, Husheng Ding, Anu Jain, Jane A Peterson, Taewook Kang, Ganesh P Pujari, Jennifer L Tomlinson, Rohit Budhraja, Roman M Zenka, Nagarajan Kannan, Benjamin R Kipp, Surendra Dasari, Alexandre Gaspar-Maia, Rory L Smoot, Richard K Kandasamy, Akhilesh Pandey.

The rapid evolution of mass spectrometry-based single-cell proteomics now enables the cataloging of several thousand proteins from single cells. We investigated whether we could discover cellular heterogeneity beyond proteome, encompassing post-translational modifications (PTM), protein-protein interaction, and variants. By optimizing the mass spectrometry data interpretation strategy to enable the detection of PTMs and variants, we have generated a high-definition dataset of single-cell and nuclear proteomic-states. The data demonstrate the heterogeneity of cell-states and signaling dependencies at the single-cell level and reveal epigenetic drug-induced changes in single nuclei. This approach enables the exploration of previously uncharted single-cell and organellar proteomes revealing molecular characteristics that are inaccessible through RNA profiling.

DOI: https://doi.org/10.1038/s42003-024-06579-7
Leukemia. 2024 Jul 18.

Proteomic screening identifies PF4/Cxcl4 as a critical driver of myelofibrosis.

Daniele Capitanio, Francesca R Calledda, Vittorio Abbonante, Daniele Cattaneo, Manuela Moriggi, Bartalucci Niccolò, Cristina Bucelli, Delfina Tosi, Umberto Gianelli, Alessandro Maria Vannucchi, Alessandra Iurlo, Cecilia Gelfi, Alessandra Balduini, Alessandro Malara.

Despite increased understanding of the genomic landscape of Myeloproliferative Neoplasms (MPNs), the pathological mechanisms underlying abnormal megakaryocyte (Mk)-stromal crosstalk and fibrotic progression in MPNs remain unclear. We conducted mass spectrometry-based proteomics on mice with Romiplostim-dependent myelofibrosis to reveal alterations in signaling pathways and protein changes in Mks, platelets, and bone marrow (BM) cells. The chemokine Platelet Factor 4 (PF4)/Cxcl4 was up-regulated in all proteomes and increased in plasma and BM fluids of fibrotic mice. High TPO concentrations sustained in vitro PF4 synthesis and secretion in cultured Mks, while Ruxolitinib restrains the abnormal PF4 expression in vivo. We discovered that PF4 is rapidly internalized by stromal cells through surface glycosaminoglycans (GAGs) to promote myofibroblast differentiation. Cxcl4 gene silencing in Mks mitigated the profibrotic phenotype of stromal cells in TPO-saturated co-culture conditions. Consistently, extensive stromal PF4 uptake and altered GAGs deposition were detected in Romiplostim-treated, JAK2V617F mice and BM biopsies of MPN patients. BM PF4 levels and Mk/platelet CXCL4 expression were elevated in patients, exclusively in overt fibrosis. Finally, pharmacological inhibition of GAGs ameliorated in vivo fibrosis in Romiplostim-treated mice. Thus, our findings highlight the critical role of PF4 in the fibrosis progression of MPNs and substantiate the potential therapeutic strategy of neutralizing PF4-GAGs interaction.

DOI: https://doi.org/10.1038/s41375-024-02354-z
Nat Commun. 2024 Jul 16. 15(1): 5967

CryoET reveals actin filaments within platelet microtubules.

Chisato Tsuji, Marston Bradshaw, Megan F Allen, Molly L Jackson, Judith Mantell, Ufuk Borucu, Alastair W Poole, Paul Verkade, Ingeborg Hers, Danielle M Paul, Mark P Dodding.

Crosstalk between the actin and microtubule cytoskeletons is important for many cellular processes. Recent studies have shown that microtubules and F-actin can assemble to form a composite structure where F-actin occupies the microtubule lumen. Whether these cytoskeletal hybrids exist in physiological settings and how they are formed is unclear. Here, we show that the short-crossover Class I actin filament previously identified inside microtubules in human HAP1 cells is cofilin-bound F-actin. Lumenal F-actin can be reconstituted in vitro, but cofilin is not essential. Moreover, actin filaments with both cofilin-bound and canonical morphologies reside within human platelet microtubules under physiological conditions. We propose that stress placed upon the microtubule network during motor-driven microtubule looping and sliding may facilitate the incorporation of actin into microtubules.

DOI: https://doi.org/10.1038/s41467-024-50424-8
Adv Exp Med Biol. 2024 ;1459 3-29

MYB: A Key Transcription Factor in the Hematopoietic System Subject to Many Levels of Control.

Roza Berhanu Lemma, Bettina Maria Fuglerud, Jon Frampton, Odd Stokke Gabrielsen.

  MYB is a master regulator and pioneer factor highly expressed in hematopoietic progenitor cells (HPCs) where it contributes to the reprogramming processes operating during hematopoietic development. MYB plays a complex role being involved in several lineages of the hematopoietic system. At the molecular level, the MYB gene is subject to intricate regulation at many levels through several enhancer and promoter elements, through transcriptional elongation control, as well as post-transcriptional regulation. The protein is modulated by post-translational modifications (PTMs) such as SUMOylation restricting the expression of its downstream targets. Together with a range of interaction partners, cooperating transcription factors (TFs) and epigenetic regulators, MYB orchestrates a fine-tuned symphony of genes expressed during various stages of haematopoiesis. At the same time, the complex MYB system is vulnerable, being a target for unbalanced control and cancer development.

Keywords:  Enhancer; Epigenetic regulator; MYB; Pioneer factor; SUMO; Target genes; Transcription factor; c-Myb

DOI:  https://doi.org/10.1007/978-3-031-62731-6_1
Nat Commun. 2024 Jul 13. 15(1): 5888

DropBlot: single-cell western blotting of chemically fixed cancer cells.

Yang Liu, Amy E Herr.

Archived patient-derived tissue specimens play a central role in understanding disease and developing therapies. To address specificity and sensitivity shortcomings of existing single-cell resolution proteoform analysis tools, we introduce a hybrid microfluidic platform (DropBlot) designed for proteoform analyses in chemically fixed single cells. DropBlot serially integrates droplet-based encapsulation and lysis of single fixed cells, with on-chip microwell-based antigen retrieval, with single-cell western blotting of target antigens. A water-in-oil droplet formulation withstands the harsh chemical (SDS, 6 M urea) and thermal conditions (98 °C, 1-2 hr) required for effective antigen retrieval, and supports analysis of retrieved protein targets by single-cell electrophoresis. We demonstrate protein-target retrieval from unfixed, paraformaldehyde-fixed (PFA), and methanol-fixed cells. Key protein targets (HER2, GAPDH, EpCAM, Vimentin) retrieved from PFA-fixed cells were resolved and immunoreactive. Relevant to biorepositories, DropBlot profiled targets retrieved from human-derived breast tumor specimens archived for six years, offering a workflow for single-cell protein-biomarker analysis of sparing biospecimens.

DOI: https://doi.org/10.1038/s41467-024-50046-0
Am J Hematol. 2024 Jul 17.

Frontline therapy of acute myeloid leukemia with lower intensity regimens: Where are we now and where can we go?

Jennifer Marvin-Peek, Jason S Gilbert, Daniel A Pollyea, Courtney D DiNardo.

The advent of molecularly targeted therapeutics has transformed the management of patients with acute myeloid leukemia (AML). Particularly for individuals unfit for intensive chemotherapy, lower intensity therapies (LIT) incorporating small molecules have significantly improved patient outcomes. With BCL2, IDH1, IDH2, and FLT3 inhibitors widely used for relapsed AML, combination regimens are now utilized in the frontline. Expansion of these targeted LIT combinations, along with development of novel agents including menin inhibitors, exemplifies the promise of precision medicine. Further understanding of molecular drivers of leukemic transformation and mechanisms of relapse will continue to advance frontline treatment options for patients with AML.

DOI: https://doi.org/10.1002/ajh.27434
Methods Mol Biol. 2024 ;2826 219-230

Assessing the Survival of Human Plasma Cells Ex Vivo.

Jack Polmear, Anne L Fletcher, Kim L Good-Jacobson.

  One way memory B cells provide protection is by rapidly differentiating into plasma cells. Plasma cells are vital in providing long-term protection against pathogens; however, they can also be detrimental to health in the case of antibody-mediated autoimmunity. Therefore, compounds which modulate the survival of plasma cells have been of interest for therapeutic intervention. Investigation of ex vivo plasma cell survival has previously been limited by the low frequency of plasma cells in the blood. Here we describe a novel ex vivo culture system that only requires 3000-5000 cells per condition. This method permits the assessment of human plasma cell survival derived from blood and can assess the impact of small molecule inhibitors on plasma cell viability.

Keywords:  Ex vivo culture; Human; Plasma cell; Small molecule inhibitor; Survival

DOI:  https://doi.org/10.1007/978-1-0716-3950-4_16
Anticancer Drugs. 2024 Jul 22.

Interference with PLA2G16 promotes cell cycle arrest and apoptosis and inhibits the reprogramming of glucose metabolism in multiple myeloma cells by modulating the Hippo/YAP signaling pathway.

Hongyan Li, Yi Zhang, Xiaoyu Mou, Bo Huang, Xiaoqiang Fan.

Multiple myeloma, which is a clonal plasma cell tumor, derives from a postmitotic lymphoid B-cell lineage and remains untreatable. Group XVI phospholipase A2 (PLA2G16) can either be a tumor suppressor or an oncogene in different types of cancer. This study was intended to explore the role of PLA2G16 in multiple myeloma and to reveal the reaction mechanism. The mRNA and protein expressions of PLA2G16 in human bone marrow stromal cell line HS-5 and multiple myeloma cells were assessed using reverse transcription-quantitative PCR and western blot. The transfection efficacy of sh-PLA2G16 and oe-YAP was examined using reverse transcription-quantitative PCR and western blot. Through cell counting kit-8 assay and 5-ethynyl-2'- deoxyuridine staining, multiple myeloma cell viability and proliferation were detected. Flow cytometry was used to measure cell apoptosis and cell cycle distribution. Oxygen consumption rate, the activities of mitochondrial respiratory chain complexes I-V, and the activity of caspase-3 were estimated with Seahorse XF24 analyzer, oxidative phosphorylation activity assay kit, and caspase-3 assay kit, respectively. Lactate production and glucose consumption were evaluated usingcorresponding assay kits. Western blot was employed to meaure proteins associated with cell cycle, glycolysis, pentose phosphate pathway as well as Hippo/YAP signaling pathway. In this study, PLA2G16 expression was greatly increased in multiple myeloma cells and PLA2G16 silence inhibited cell proliferation, promoted cell apoptosis, facilitated cell cycle arrest, and suppressed the reprogramming of glucose metabolism in multiple myeloma. It was also identified that PLA2G16 depletion inhibited the Hippo/YAP signaling pathway. Further experiments revealed that the overexpression of YAP partially reversed the inhibitory effects of PLA2G16 silence on multiple myeloma cell malignant development and the reprogramming of glucose metabolism. Collectively, PLA2G16 silence impeded multiple myeloma progression and inhibited glucose metabolism reprogramming by blocking the Hippo/YAP signaling pathway.

DOI: https://doi.org/10.1097/CAD.0000000000001642
J Biol Chem. 2024 Jul 11. pii: S0021-9258(24)02067-2. [Epub ahead of print] 107566

MLL-AF9 regulates transcriptional initiation in mixed lineage leukemic cells.

Zimei Yang, Ge Zhang, Ruoyu Zhao, Tian Tian, Junhong Zhi, Gang Wei, Robert G Roeder, Lili Jing, Ming Yu.

  MLL-fusion proteins (MLL-FPs) are believed to maintain gene activation and induce mixed lineage leukemia (MLL) through aberrantly stimulating transcriptional elongation, but the underlying mechanisms are incompletely understood. Here we show that both MLL1 and AF9, one of the major fusion partners of MLL1, mainly occupy promoters and distal intergenic regions, exhibiting chromatin occupancy patterns resembling that of RNA polymerase II (Pol II) in HEL, a human cell line without MLL1 arrangement (MLLr). MLL1 and AF9 only co-regulate over a dozen genes despite of their co-occupancy on thousands of genes. They do not interact with each other, and their chromatin occupancy is also independent of each other. Moreover, AF9 deficiency in HEL cells decreases global TBP occupancy while decreases CDK9 occupancy on a small number of genes, suggesting an accessory role of AF9 in CDK9 recruitment and a possible major role in transcriptional initiation via initiation factor recruitment. Importantly, MLL1 and MLL-AF9 occupy promoters and distal intergenic regions, exhibiting identical chromatin occupancy patterns in MLL cells, and MLL-AF9 deficiency decreased occupancy of TBP and TFIIE on major target genes of MLL-AF9 in iMA9, a murine acute myeloid leukemia (AML) cell line inducibly expressing MLL-AF9, suggesting that it can also regulate initiation. These results suggest that there is no difference between MLL1 and MLL-AF9 with respect to location and size of occupancy sites, contrary to what people have believed, and that MLL-AF9 may also regulate transcriptional initiation in addition to widely-believed elongation.

Keywords:  AF9; MLL-AF9; MLL1; initiation; mixed lineage leukemia; super elongation complex

DOI:  https://doi.org/10.1016/j.jbc.2024.107566
bioRxiv. 2024 Jul 02. pii: 2024.06.27.601046. [Epub ahead of print]

circPCMTD1 : A protein-coding circular RNA that regulates DNA damage response in BCR/ABL -positive leukemias.

Dimitrios Papaioannou, Amog P Urs, Rémi Buisson, Andreas Petri, Rohan Kulkarni, Deedra Nicolet, Lauren Woodward, Chinmayee Goda, Krzysztof Mrózek, Gregory K Behbehani, Sakari Kauppinen, Ann-Kathrin Eisfeld, Iannis Aifantis, Guramrit Singh, Adrienne M Dorrance, Ramiro Garzon.

Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of the circPCMTD1 transcript in acute leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR-ABL translocations. Mass cytometry experiments identified the aberrant activation of the DNA damage response as an early downstream event of circPCMTD1 depletion. In in vivo experiments, circPCMTD1 targeting prolonged the survival of mice engrafted with leukemic blasts harboring the Philadelphia chromosome. Mechanistically, we found that circPCMTD1 was enriched in the cytoplasm and associated with the ribosomes of the leukemic cells. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 could generate a peptide product. The circPCMTD 1-derived peptide interacted with proteins of the BTR complex and enhanced BTR complex formation, thereby increasing tolerance to genotoxic stress.

DOI: https://doi.org/10.1101/2024.06.27.601046
Biochem Biophys Res Commun. 2024 Jul 14. pii: S0006-291X(24)00933-1. [Epub ahead of print]731 150397

The erlin1/erlin2 complex binds to and stabilizes phosphatidylinositol 3-phosphate and regulates autophagy.

Fanghui Hua, Caden G Bonzerato, Katherine R Keller, Dandan Guo, Juntao Luo, Richard J H Wojcikiewicz.

  The erlin1/erlin2 (E1/E2) complex is an endoplasmic reticulum membrane-located assemblage of the proteins erlin1 and erlin2. Here, we demonstrate direct and selective binding of phosphatidylinositol 3-phosphate (PI(3)P) to recombinant erlins and that disruption or deletion of the E1/E2 complex reduces HeLa cell PI(3)P levels by ∼50 %. This reduction correlated with a decrease in autophagic flux, with no effect on the endocytic pathway, and was not due to reduced VPS34 kinase activity, which is critical for maintaining steady-state PI(3)P levels. Pharmacological inhibition of VPS34 and suppression of PI(3)P levels caused a similar reduction in autophagic flux. Overall, these data indicate that by binding to PI(3)P, the E1/E2 complex plays an important role in maintaining the steady-state levels of PI(3)P and, thus, sustains some key PI(3)P-dependent processes, e.g., autophagy.

Keywords:  Autophagy; Endocytosis; Erlin complex; Lipid metabolism; Phosphatidylinositol 3-phosphate

DOI:  https://doi.org/10.1016/j.bbrc.2024.150397
Heliyon. 2024 Jun 30. 10(12): e33091

Pathways to therapy resistance: The sheltering effect of the bone marrow microenvironment to multiple myeloma cells.

Kuntal Bhowmick, Max von Suskil, Omar S Al-Odat, Weam Othman Elbezanti, Subash C Jonnalagadda, Tulin Budak-Alpdogan, Manoj K Pandey.

Multiple Myeloma (MM) is a malignant expansion of plasma cells in the bone marrow (BM), resulting in a disease characterized by symptoms of end organ damage from light chain secretion, crowding of the BM, and bone lesions. Although the past two decades have been characterized by numerous novel therapies emerging, the disease remains incurable due to intrinsic or acquired drug resistance. A major player in MM's drug resistance arises from its intimate relationship with the BM microenvironment (BMME). Through stress-inducing conditions, soluble messengers, and physical adhesion to BM elements, the BMME activates numerous pathways in the myeloma cell. This not only propagates myeloma progression through survival and growth signals, but also specific mechanisms to circumvent therapeutic actions. In this review, we provide an overview of the BMME, the role of individual components in MM survival, and various therapy-specific resistance mechanisms reported in the literature.

DOI: https://doi.org/10.1016/j.heliyon.2024.e33091
Cancer Res Commun. 2024 Jul 15.

FLT3/CD99 Bispecific antibody-based nanoparticles (BiAbs) for Acute Myeloid Leukemia.

Atham Ali, Alvin Phan, Vijaya Vaikari, Mincheol Park, Mateusz Pospiech, Ryan Chu, Yiting Meng, John A MacKay, Houda Alachkar.

CD99 is a receptor that is significantly upregulated in AML. FLT3-ITD AML exhibits even higher levels of CD99 expression. Our group previously employed a novel peptide platform technology called elastin-like polypeptides and fused it with single-chain antibodies (scFv) capable of binding to FLT3 (FLT3-A192), or CD99 (CD99-A192). Targeting either FLT3 or CD99 using FLT3-A192 or CD99-A192 led to AML cell death and reduced leukemia burden in AML mouse models. Here, we report on the development of a novel co-assembled construct that is capable to binding both CD99 and FLT3 and the antileukemia activity of the bispecific construct in FLT3-ITD AML preclinical models. This dual-targeting Co-Assembled formulation exhibits cytotoxic effects on AML cells (AML cell lines and primary blasts) and reduced leukemia burden and prolonged survival in FLT3-ITD AML mouse models. Altogether, this study demonstrates the potential of an innovative therapeutic strategy that targets both FLT3 and CD99 in FLT3-ITD AML.

DOI: https://doi.org/10.1158/2767-9764.CRC-24-0096
J Extracell Vesicles. 2024 Jul;13(7): e12449

Cancer EV stimulate endothelial glycolysis to fuel protein synthesis via mTOR and AMPKα activation.

Joël E J Beaumont, Lydie M O Barbeau, Jinzhe Ju, Kim G Savelkouls, Freek G Bouwman, Marijke I Zonneveld, Annelies Bronckaers, Kim R Kampen, Tom G H Keulers, Kasper M A Rouschop.

  Hypoxia is a common feature of solid tumours and activates adaptation mechanisms in cancer cells that induce therapy resistance and has profound effects on cellular metabolism. As such, hypoxia is an important contributor to cancer progression and is associated with a poor prognosis. Metabolic alterations in cells within the tumour microenvironment support tumour growth via, amongst others, the suppression of immune reactions and the induction of angiogenesis. Recently, extracellular vesicles (EV) have emerged as important mediators of intercellular communication in support of cancer progression. Previously, we demonstrated the pro-angiogenic properties of hypoxic cancer cell derived EV. In this study, we investigate how (hypoxic) cancer cell derived EV mediate their effects. We demonstrate that cancer derived EV regulate cellular metabolism and protein synthesis in acceptor cells through increased activation of mTOR and AMPKα. Using metabolic tracer experiments, we demonstrate that EV stimulate glucose uptake in endothelial cells to fuel amino acid synthesis and stimulate amino acid uptake to increase protein synthesis. Despite alterations in cargo, we show that the effect of cancer derived EV on recipient cells is primarily determined by the EV producing cancer cell type rather than its oxygenation status.

Keywords:  angiogenesis; extracellular vesicles; hypoxia; metabolism

DOI:  https://doi.org/10.1002/jev2.12449
J Cell Biol. 2024 Aug 05. pii: e202311153. [Epub ahead of print]223(8):

Centrosome age breaks spindle size symmetry even in cells thought to divide symmetrically.

Alexandre Thomas, Patrick Meraldi.

Centrosomes are the main microtubule-organizing centers in animal cells. Due to the semiconservative nature of centrosome duplication, the two centrosomes differ in age. In asymmetric stem cell divisions, centrosome age can induce an asymmetry in half-spindle lengths. However, whether centrosome age affects the symmetry of the two half-spindles in tissue culture cells thought to divide symmetrically is unknown. Here, we show that in human epithelial and fibroblastic cell lines centrosome age imposes a mild spindle asymmetry that leads to asymmetric cell daughter sizes. At the mechanistic level, we show that this asymmetry depends on a cenexin-bound pool of the mitotic kinase Plk1, which favors the preferential accumulation on old centrosomes of the microtubule nucleation-organizing proteins pericentrin, γ-tubulin, and Cdk5Rap2, and microtubule regulators TPX2 and ch-TOG. Consistently, we find that old centrosomes have a higher microtubule nucleation capacity. We postulate that centrosome age breaks spindle size symmetry via microtubule nucleation even in cells thought to divide symmetrically.

DOI: https://doi.org/10.1083/jcb.202311153
Nat Chem. 2024 Jul 19.

Protein semisynthesis reveals plasticity in HECT E3 ubiquitin ligase mechanisms.

Hanjie Jiang, Bryant D Miller, Thibault Viennet, Hyojeon Kim, Kwangwoon Lee, Haribabu Arthanari, Philip A Cole.

Lys ubiquitination is catalysed by E3 ubiquitin ligases and is central to the regulation of protein stability and cell signalling in normal and disease states. There are gaps in our understanding of E3 mechanisms, and here we use protein semisynthesis, chemical rescue, microscale thermophoresis and other biochemical approaches to dissect the role of catalytic base/acid function and conformational interconversion in HECT-domain E3 catalysis. We demonstrate that there is plasticity in the use of the terminal side chain or backbone carboxylate for proton transfer in HECT E3 ubiquitin ligase reactions, with yeast Rsp5 orthologues appearing to be possible evolutionary intermediates. We also show that the HECT-domain ubiquitin covalent intermediate appears to eject the E2 conjugating enzyme, promoting catalytic turnover. These findings provide key mechanistic insights into how protein ubiquitination occurs and provide a framework for understanding E3 functions and regulation.

DOI: https://doi.org/10.1038/s41557-024-01576-z
Blood Cancer J. 2024 Jul 19. 14(1): 117

Genomic and immune determinants of resistance to daratumumab-based therapy in relapsed refractory multiple myeloma.

Bachisio Ziccheddu, Claudia Giannotta, Mattia D'Agostino, Giuseppe Bertuglia, Elona Saraci, Stefania Oliva, Elisa Genuardi, Marios Papadimitriou, Benjamin Diamond, Paolo Corradini, David Coffey, Ola Landgren, Niccolò Bolli, Benedetto Bruno, Mario Boccadoro, Massimo Massaia, Francesco Maura, Alessandra Larocca.

Targeted immunotherapy combinations, including the anti-CD38 monoclonal antibody (MoAb) daratumumab, have shown promising results in patients with relapsed/refractory multiple myeloma (RRMM), leading to a considerable increase in progression-free survival. However, a large fraction of patients inevitably relapse. To understand this, we investigated 32 relapsed MM patients treated with daratumumab, lenalidomide, and dexamethasone (Dara-Rd; NCT03848676). We conducted an integrated analysis using whole-genome sequencing (WGS) and flow cytometry in patients with RRMM. WGS before and after treatment pinpointed genomic drivers associated with early progression, including RPL5 loss, APOBEC mutagenesis, and gain of function structural variants involving MYC and chromothripsis. Flow cytometry on 202 blood samples, collected every 3 months until progression for 31 patients, revealed distinct immune changes significantly impacting clinical outcomes. Progressing patients exhibited significant depletion of CD38-positive NK cells, persistence of T-cell exhaustion, and reduced depletion of regulatory T cells over time. These findings underscore the influence of immune composition and daratumumab-induced immune changes in promoting MM resistance. Integrating genomics and flow cytometry unveiled associations between adverse genomic features and immune patterns. Overall, this study sheds light on the intricate interplay between genomic complexity and the immune microenvironment driving resistance to Dara-Rd in patients with RRMM.

DOI: https://doi.org/10.1038/s41408-024-01096-6
Nature. 2024 Jul 17.

Clonal inactivation of TERT impairs stem cell competition.

Kazuteru Hasegawa, Yang Zhao, Alina Garbuzov, M Ryan Corces, Patrick Neuhöfer, Victoria M Gillespie, Peggie Cheung, Julia A Belk, Yung-Hsin Huang, Yuning Wei, Lu Chen, Howard Y Chang, Steven E Artandi.

Telomerase is intimately associated with stem cells and cancer, because it catalytically elongates telomeres-nucleoprotein caps that protect chromosome ends1. Overexpression of telomerase reverse transcriptase (TERT) enhances the proliferation of cells in a telomere-independent manner2-8, but so far, loss-of-function studies have provided no evidence that TERT has a direct role in stem cell function. In many tissues, homeostasis is shaped by stem cell competition, a process in which stem cells compete on the basis of inherent fitness. Here we show that conditional deletion of Tert in the spermatogonial stem cell (SSC)-containing population in mice markedly impairs competitive clone formation. Using lineage tracing from the Tert locus, we find that TERT-expressing SSCs yield long-lived clones, but that clonal inactivation of TERT promotes stem cell differentiation and a genome-wide reduction in open chromatin. This role for TERT in competitive clone formation occurs independently of both its reverse transcriptase activity and the canonical telomerase complex. Inactivation of TERT causes reduced activity of the MYC oncogene, and transgenic expression of MYC in the TERT-deleted pool of SSCs efficiently rescues clone formation. Together, these data reveal a catalytic-activity-independent requirement for TERT in enhancing stem cell competition, uncover a genetic connection between TERT and MYC and suggest that a selective advantage for stem cells with high levels of TERT contributes to telomere elongation in the male germline during homeostasis and ageing.

DOI: https://doi.org/10.1038/s41586-024-07700-w
Res Sq. 2024 Jul 03. pii: rs.3.rs-4590410. [Epub ahead of print]

Benchmarking and Automating the Biotinylation Proteomics Workflow.

Ling Hao, Haorong Li, Noah Smeriglio, Jiawei Ni, Yan Wang, Shiori Sekine.

Protein biotinylation has been widely used in biotechnology with various labeling and enrichment strategies. However, different enrichment strategies have not been systematically evaluated due to the lack of a benchmarking model for fair comparison. Most biotinylation proteomics workflows suffer from lengthy experimental steps, non-specific bindings, limited throughput, and experimental variability. To address these challenges, we designed a two-proteome model, where biotinylated yeast proteins were spiked in unlabeled human proteins, allowing us to distinguish true enrichment from non-specific bindings. Using this benchmarking model, we compared common biotinylation proteomics methods and provided practical selection guidelines. We significantly optimized and shortened sample preparation from 3 days to 9 hours, enabling fully-automated 96-well plate sample processing. Next, we applied this optimized and automated workflow for proximity labeling to investigate the intricate interplay between mitochondria and lysosomes in living cells under both healthy state and mitochondrial damage. Our results suggested a time-dependent proteome remodeling and dynamic translocation within mitochondria and between mitochondria and lysosomes upon mitochondrial damage. This newly established benchmarking model and the fully-automated 9-hour workflow can be readily applied to the broad fields of protein biotinylation to study protein interaction and organelle dynamics.

DOI: https://doi.org/10.21203/rs.3.rs-4590410/v1
Adv Mater. 2024 Jul 17. e2403952

Long-Term Culture of Human Pluripotent Stem Cells in Xeno-Free Condition Using Functional Polymer Films.

Younghak Cho, Hana Lee, Wonji Jeong, Kwang Bo Jung, Sun Young Lee, Seonghyeon Park, Jemin Yeun, Ohman Kwon, Jin Gyeong Son, Tae Geol Lee, Mi-Young Son, Sung Gap Im.

  Human pluripotent stem cells (hPSCs), encompassing human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold immense potential in regenerative medicine, offering new opportunities for personalized cell therapies. However, their clinical translation is hindered by the inevitable reliance on xenogeneic components in culture environments. This study addresses this challenge by engineering a fully synthetic, xeno-free culture substrate, whose surface composition is tailored systematically for xeno-free culture of hPSCs. A functional polymer surface, pGC2 (poly(glycidyl methacrylate-grafting-guanidine-co-carboxylic acrylate)), offers excellent cell-adhesive properties as well as non-cytotoxicity, enabling robust hESCs and hiPSCs growth while presenting cost-competitiveness and scalability over Matrigel. This investigation includes comprehensive evaluations of pGC2 across diverse experimental conditions, demonstrating its wide adaptability with various pluripotent stem cell lines, culture media, and substrates. Crucially, pGC2 supports long-term hESCs and hiPSCs expansion, up to ten passages without compromising their stemness and pluripotency. Notably, this study is the first to confirm an identical proteomic profile after ten passages of xeno-free cultivation of hiPSCs on a polymeric substrate compared to Matrigel. The innovative substrate bridges the gap between laboratory research and clinical translation, offering a new promising avenue for advancing stem cell-based therapies.

Keywords:  cell therapeutics; human pluripotent stem cells; initiated chemical vapor deposition; long‐term culture; pluripotency; stemness; xeno‐free culture

DOI:  https://doi.org/10.1002/adma.202403952
Anal Chem. 2024 Jul 16.

A Hypochlorite-Activated Theranostic Prodrug for Selective Imaging of High Myeloperoxidase Expression Acute Myeloid Leukemia Cells and Drug Release.

Yuting Wang, Xiaoyu Cheng, Nan Yin, Mingxiu Wang, Guixin Qin, Jiali Tang, Yinlong Zhang, Qingling Xu.

Acute myeloid leukemia (AML) is a fatal hematologic disease. Diagnosis and proper treatment are important for prognosis. High myeloperoxidase (MPO) expression AML cells are characterized with high levels of hypochlorite (ClO-). In this study, we report a ClO--activated theranostic agent, FNC, for AML therapy. FNC responds to ClO- specifically in high MPO expression AML cells, resulting in bright fluorescence and chlorambucil release. FNC can be used to quickly distinguish high MPO expression AML cells from other cells, including low MPO expression leukemia and activated inflammatory cells. FNC exhibits selective toxicity to highly MPO expression AML cells and can efficiently inhibit tumor growth. Meanwhile, FNC can be used to indicate differentiation through the detection of ClO-.

DOI: https://doi.org/10.1021/acs.analchem.4c00273