bims-scepro 2024-06-23 papers

bims-scepro

Biomed News

on Stem cell proteostasis

Issue of 2024–06–23
33 papers selected by
William Grey, University of York

Gelatin maleimide microgels for hematopoietic progenitor cell encapsulation.
NEUROBEACHIN regulates hematopoietic progenitor differentiation and survival by modulating NOTCH activity.
Low Kit expression identifies Hematopoietic Stem Cell subsets with enhanced lymphoid potential in mice and humans.
Wnt signaling couples G2 phase control with differentiation during hematopoiesis in Drosophila.
Systemic knockout of Tmem175 results in aberrant differentiation but no effect on hematopoietic reconstitution.
Nutrient-sensitizing drug repurposing screen identifies lomerizine as a mitochondrial metabolism inhibitor of chronic myeloid leukemia.
From Marrow to Bone and Fat: Exploring the Multifaceted Roles of Leptin Receptor Positive Bone Marrow Mesenchymal Stromal Cells.
Improvement of multilineage hematopoiesis in hematopoietic stem cell-transferred c-kit mutant NOG-EXL humanized mice.
HIF-2α Inhibition Disrupts Leukemia Stem Cell Metabolism and Impairs Vascular Microenvironment to Enhance Chronic Myeloid Leukemia Treatment.
Intracellular pH dynamics respond to microenvironment stiffening and mediate vasculogenic mimicry through β-catenin.
Hematopoietic stem cell division is governed by distinct RUNX1 binding partners.
Distinct Responses to Menin Inhibition and Synergy with DOT1L Inhibition in KMT2A-Rearranged Acute Lymphoblastic and Myeloid Leukemia.
CYR61 Acts as an Intracellular Microtubule-Associated Protein and Coordinates Mitotic Progression via PLK1-FBW7 Pathway.
GATA2 heterozygosity causes an epigenetic feedback mechanism resulting in myeloid and erythroid dysplasia.
Calorie restriction and calorie-restriction mimetics activate chaperone-mediated autophagy.
Extracellular matrices of stromal cell subtypes regulate phenotype and contribute to the stromal microenvironment in vivo.
C-terminally phosphorylated p27 activates self-renewal driver genes to program cancer stem cell expansion, mammary hyperplasia and cancer.
Molecular Changes in Immunological Characteristics of Bone Marrow Multipotent Mesenchymal Stromal Cells in Lymphoid Neoplasia.
Step by step analysis on gene datasets of growth phases in hematopoietic stem cells.
Protein arginine methyltransferase 2 controls inflammatory signaling in acute myeloid leukemia.
SOD1 is a synthetic-lethal target in PPM1D-mutant leukemia cells.
Pin1-catalyzed conformational regulation after phosphorylation: A distinct checkpoint in cell signaling and drug discovery.
Single-cell proteomics reveals decreased abundance of proteostasis and meiosis proteins in advanced maternal age oocytes.
Astrocytes control quiescent NSC reactivation via GPCR signaling-mediated F-actin remodeling.
Synergistic effects of the KDM4C inhibitor SD70 and the menin inhibitor MI-503 against MLL::AF9-driven acute myeloid leukaemia.
UFMylation: An integral post-translational modification for the regulation of proteostasis and cellular functions.
Single-Cell Proteomics Analysis with Tecan Uno and SCREEN Workflow.
Azab AK, Quang P, Azab F, et al. P-selectin glycoprotein ligand regulates the interaction of multiple myeloma cells with the bone marrow microenvironment. Blood. 2012;119(6):1468-1478.
Label-Free Sample Preparation for Single-Cell Proteomics.
Spatial Proteomics of Single Hepatocytes with Multiplexed Data-Independent Acquisition (mDIA).
Oncogenic Calreticulin Induces Immune Escape by Stimulating TGF-β Expression and Regulatory T Cell Expansion in the Bone Marrow Microenvironment.
Expression of modified FcγRI enables myeloid cells to elicit robust tumor-specific cytotoxicity.
Recent Advances in Immune-Based Therapies for Acute Myeloid Leukemia.

J Biomed Mater Res A. 2024 Jun 19.

Gelatin maleimide microgels for hematopoietic progenitor cell encapsulation.

Gunnar B Thompson, Aidan E Gilchrist, Vincent M Lam, Alison C Nunes, Brittany A Payan, Ana Mora-Boza, Julio F Serrano, Andrés J García, Brendan A C Harley.

  Hematopoietic stem cells (HSCs) are the apical cells of the hematopoietic system, giving rise to cells of the blood and lymph lineages. HSCs reside primarily within bone marrow niches that contain matrix and cell-derived signals that help inform stem cell fate. Aspects of the bone marrow microenvironment have been captured in vitro by encapsulating cells within hydrogel matrices that mimic native mechanical and biochemical properties. Hydrogel microparticles, or microgels, are increasingly being used to assemble granular biomaterials for cell culture and noninvasive delivery applications. Here, we report the optimization of a gelatin maleimide hydrogel system to create monodisperse gelatin microgels via a flow-focusing microfluidic process. We report characteristic hydrogel stiffness, stability, and swelling characteristics as well as encapsulation of murine hematopoietic stem and progenitor cells, and mesenchymal stem cells within microgels. Microgels support cell viability, confirming compatibility of the microfluidic encapsulation process with these sensitive bone marrow cell populations. Overall, this work presents a microgel-based gelatin maleimide hydrogel as a foundation for future development of a multicellular artificial bone marrow culture system.

Keywords:  bone marrow; gelatin; hematopoietic stem cell; microgel

DOI:  https://doi.org/10.1002/jbm.a.37765
Blood Adv. 2024 Jun 21. pii: bloodadvances.2023012426. [Epub ahead of print]

NEUROBEACHIN regulates hematopoietic progenitor differentiation and survival by modulating NOTCH activity.

Miguel Ganuza Fernandez, Antonio Morales-Hernandez, Alanna Van Huizen, Ashley Chabot, Trent Hall, Claire Caprio, David Finkelstein, Shannon L McKinney Freeman.

Hematopoietic stem cells (HSCs) can generate all blood cells. This ability is exploited in HSC transplantation (HSCT) to treat hematologic disease. A clear understanding of the molecular mechanisms that regulate HSCT is necessary to continue improving transplant protocols. We identified the BEACH-domain containing protein (BDCP), NEUROBEACHIN (NBEA), as a putative regulator of HSCT. Here, we demonstrated that NBEA and related BDCPs, including LRBA, NBEAL1 and LYST, are required during HSCT to efficiently reconstitute the hematopoietic system of lethally irradiated mice. Nbea knockdown in mouse HSCs induced apoptosis and a differentiation block post-transplantation. Nbea deficiency in hematopoietic progenitor cells perturbed the expression of genes implicated in vesicle trafficking and led to changes in NOTCH receptor localization. This resulted in perturbation of the NOTCH transcriptional program, which is required for efficient HSC engraftment. In sum, our findings reveal a novel role for NBEA in the control of NOTCH receptor turnover in hematopoietic cells and supports a model where BDCP regulated vesicle trafficking is required for efficient HSCT.

DOI: https://doi.org/10.1182/bloodadvances.2023012426
bioRxiv. 2024 Jun 09. pii: 2024.06.06.597775. [Epub ahead of print]

Low Kit expression identifies Hematopoietic Stem Cell subsets with enhanced lymphoid potential in mice and humans.

Harold K Elias, Sneha Mitra, Marina B da Silva, Adhithi Rajagopalan, Brianna Gipson, Nicole Lee, Anastasia I Kousa, Mohamed A E Ali, Simon Grassman, Xiaoqun Zhang, Susan DeWolf, Melody Smith, Hana Andrlova, Kimon V Argyropoulos, Roshan Sharma, Teng Fei, Joseph C Sun, Cynthia E Dunbar, Christopher Y Park, Christina S Leslie, Avinash Bhandoola, Marcel R M van den Brink.

Hematopoietic stem cells (HSCs) with multilineage potential are critical for effective T cell reconstitution and restoration of the adaptive immune system after allogeneic Hematopoietic Cell Transplantation (allo-HCT). Our current understanding of lymphoid-poised HSC subsets is limited due to a lack of reliable markers. Through comprehensive molecular profiling of HSCs, we identify Kit lo HSCs-a subset with enhanced lymphoid potential, which declines with age. Using a preclinical allo-HCT model, we demonstrate that Kit lo HSCs exhibit superior lymphoid progenitor output, enhanced thymic recovery, and T-cell reconstitution resulting in improved T cell responses to infection post-HCT. Furthermore, Kit lo HSCs with augmented BM lymphopoiesis mitigate age-associated thymic alterations, thus enhancing T-cell recovery in middle-aged hosts. Chromatin profiling revealed that Kit lo HSCs exhibit higher activity of lymphoid-specifying transcription factors (TFs), including Zbtb1 . Deletion of Zbtb1 in Kit lo HSCs, diminished their T-cell potential, while reinstating Zbtb1 in megakaryocytic-biased Kit hi HSCs rescued T-cell potential, in vitro and in vivo . Finally, we discover an analogous Kit lo HSC subset with enhanced lymphoid potential in human bone marrow. Our results demonstrate that Kit lo HSCs identify HSC subsets with enhanced lymphoid potential mediated by an underlying epigenetic program.

DOI: https://doi.org/10.1101/2024.06.06.597775
Dev Cell. 2024 Jun 06. pii: S1534-5807(24)00341-1. [Epub ahead of print]

Wnt signaling couples G2 phase control with differentiation during hematopoiesis in Drosophila.

Lauren M Goins, Juliet R Girard, Bama Charan Mondal, Sausan Buran, Chloe C Su, Ruby Tang, Titash Biswas, Jessica A Kissi, Utpal Banerjee.

  During homeostasis, a critical balance is maintained between myeloid-like progenitors and their differentiated progeny, which function to mitigate stress and innate immune challenges. The molecular mechanisms that help achieve this balance are not fully understood. Using genetic dissection in Drosophila, we show that a Wnt6/EGFR-signaling network simultaneously controls progenitor growth, proliferation, and differentiation. Unlike G1-quiescence of stem cells, hematopoietic progenitors are blocked in G2 phase by a β-catenin-independent (Wnt/STOP) Wnt6 pathway that restricts Cdc25 nuclear entry and promotes cell growth. Canonical β-catenin-dependent Wnt6 signaling is spatially confined to mature progenitors through localized activation of the tyrosine kinases EGFR and Abelson kinase (Abl), which promote nuclear entry of β-catenin and facilitate exit from G2. This strategy combines transcription-dependent and -independent forms of both Wnt6 and EGFR pathways to create a direct link between cell-cycle control and differentiation. This unique combinatorial strategy employing conserved components may underlie homeostatic balance and stress response in mammalian hematopoiesis.

Keywords:  Cdc25; Drosophila; EGFR; G2 phase; Wnt6; blood development; cell cycle; hematopoiesis; lymph gland; β-catenin

DOI:  https://doi.org/10.1016/j.devcel.2024.05.023
Stem Cell Res. 2024 Jun 10. pii: S1873-5061(24)00167-3. [Epub ahead of print]79 103469

Systemic knockout of Tmem175 results in aberrant differentiation but no effect on hematopoietic reconstitution.

Jingjing Su, Yue Wang, Jiyuan Yao, Leimin Sun, Chunzhen Zhao, Leiming Liu, Lingling Zhang.

  Lysosomes play crucial roles in regulating cell metabolism, and K+ channels are critical for controlling various aspects of lysosomal function. Additionally, lysosomal activity is essential for maintaining the quiescence of hematopoietic stem cells (HSCs) under both steady-state and stress conditions. Tmem175 is a lysosomal potassium channel protein. To further investigate the role of K+ channels in HSCs, our study employed knockout mice to examine the function of Tmem175. Our research findings demonstrate that the deletion of Tmem175 does not disrupt the functionality of HSCs in both stable and stressed conditions, including irradiation and intraperitoneal 5-FU injections. However, we did observe that the absence of Tmem175 impairs the long-term differentiation capacity of HSCs into myeloid differentiated subpopulation cells（In this paper, it is referred to simply as M cells）in HSC transplantation test, while promoting their differentiation into T cells. This suggests that Tmem175 plays a role in the lineage differentiation of HSCs without being essential for their self-renewal or long-term regenerative capabilities.

Keywords:  Bone marrow transplantation; Hematopoietic stem cell; Lysosome; Potassium channel; Tmem175

DOI:  https://doi.org/10.1016/j.scr.2024.103469
Sci Transl Med. 2024 Jun 12. 16(751): eadi5336

Nutrient-sensitizing drug repurposing screen identifies lomerizine as a mitochondrial metabolism inhibitor of chronic myeloid leukemia.

Ahmed Khalaf, Lucie de Beauchamp, Eric Kalkman, Kevin Rattigan, Ekaterini Himonas, Joe Jones, Daniel James, Engy Shokry Abd Shokry, Mary T Scott, Karen Dunn, Saverio Tardito, Mhairi Copland, David Sumpton, Emma Shanks, G Vignir Helgason.

In chronic myeloid leukemia (CML), the persistence of leukemic stem cells (LSCs) after treatment with tyrosine kinase inhibitors (TKIs), such as imatinib, can lead to disease relapse. It is known that therapy-resistant LSCs rely on oxidative phosphorylation (OXPHOS) for their survival and that targeting mitochondrial respiration sensitizes CML LSCs to imatinib treatment. However, current OXPHOS inhibitors have demonstrated limited efficacy or have shown adverse effects in clinical trials, highlighting that identification of clinically safe oxidative pathway inhibitors is warranted. We performed a high-throughput drug repurposing screen designed to identify mitochondrial metabolism inhibitors in myeloid leukemia cells. This identified lomerizine, a US Food and Drug Administration (FDA)-approved voltage-gated Ca2+ channel blocker now used for the treatment of migraines, as one of the top hits. Transcriptome analysis revealed increased expression of voltage-gated CACNA1D and receptor-activated TRPC6 Ca2+ channels in CML LSCs (CD34+CD38-) compared with normal counterparts. This correlated with increased endoplasmic reticulum (ER) mass and increased ER and mitochondrial Ca2+ content in CML stem/progenitor cells. We demonstrate that lomerizine-mediated inhibition of Ca2+ uptake leads to ER and mitochondrial Ca2+ depletion, with similar effects seen after CACNA1D and TRPC6 knockdown. Through stable isotope-assisted metabolomics and functional assays, we observe that lomerizine treatment inhibits mitochondrial isocitrate dehydrogenase activity and mitochondrial oxidative metabolism and selectively sensitizes CML LSCs to imatinib treatment. In addition, combination treatment with imatinib and lomerizine reduced CML tumor burden, targeted CML LSCs, and extended survival in xenotransplantation model of human CML, suggesting this as a potential therapeutic strategy to prevent disease relapse in patients.

DOI: https://doi.org/10.1126/scitranslmed.adi5336
Cells. 2024 May 24. pii: 910. [Epub ahead of print]13(11):

From Marrow to Bone and Fat: Exploring the Multifaceted Roles of Leptin Receptor Positive Bone Marrow Mesenchymal Stromal Cells.

Parash Prasad, Jose A Cancelas.

  The bone marrow (BM) stromal cell microenvironment contains non-hematopoietic stromal cells called mesenchymal stromal cells (MSCs). MSCs are plastic adherent, form CFU-Fs, and give rise to osteogenic, adipogenic, chondrogenic progenitors, and most importantly provide HSC niche factor chemokine C-X-C motif ligand 12 (CXCL12) and stem cell factor (SCF). Different authors have defined different markers for mouse MSC identification like PDGFR+Sca-1+ subsets, Nestin+, or LepR+ cells. Of these, the LepR+ cells are the major source of SCF and CXCL12 in the BM microenvironment and play a major role in HSC maintenance and hematopoiesis. LepR+ cells give rise to most of the bones and BM adipocytes, further regulating the microenvironment. In adult BM, LepR+ cells are quiescent but after fracture or irradiation, they proliferate and differentiate into mesenchymal lineage osteogenic, adipogenic and/or chondrogenic cells. They also play a crucial role in the steady-state hematopoiesis process, as well as hematopoietic regeneration and the homing of hematopoietic stem cells (HSCs) after myeloablative injury and/or HSC transplantation. They line the sinusoidal cavities, maintain the trabeculae formation, and provide the space for HSC homing and retention. However, the LepR+ cell subset is heterogeneous; some subsets have higher adipogenic potential, while others express osteollineage-biased genes. Different transcription factors like Early B cell factor 3 (EBF3) or RunX2 help maintain this balance between the self-renewing and committed states, whether osteogenic or adipogenic. The study of LepR+ MSCs holds immense promise for advancing our understanding of HSC biology, tissue regeneration, metabolic disorders, and immune responses. In this review, we will discuss the origin of the BM resident LepR+ cells, different subtypes, and the role of LepR+ cells in maintaining hematopoiesis, osteogenesis, and BM adipogenesis following their multifaceted impact.

Keywords:  adipogenesis; bone marrow; hematopoiesis; leptin receptor; mesenchymal stromal cells; osteogenesis

DOI:  https://doi.org/10.3390/cells13110910
Stem Cell Res Ther. 2024 Jun 21. 15(1): 182

Improvement of multilineage hematopoiesis in hematopoietic stem cell-transferred c-kit mutant NOG-EXL humanized mice.

Ryoji Ito, Yusuke Ohno, Yunmei Mu, Yuyo Ka, Shuko Ito, Maiko Emi-Sugie, Misa Mochizuki, Kenji Kawai, Motohito Goto, Tomoyuki Ogura, Riichi Takahashi, Akira Niwa, Tatsutoshi Nakahata, Mamoru Ito.

  Human hematopoietic stem cell (HSC)-transferred humanized mice are valuable models for exploring human hematology and immunology. However, sufficient recapitulation of human hematopoiesis in mice requires large quantities of enriched human CD34+ HSCs and total-body irradiation for adequate engraftment. Recently, we generated a NOG mouse strain with a point mutation in the c-kit tyrosine kinase domain (W41 mutant; NOGW mice). In this study, we examined the ability of NOGW mice to reconstitute human hematopoietic cells. Irradiated NOGW mice exhibited high engraftment levels of human CD45+ cells in the peripheral blood, even when only 5,000-10,000 CD34+ HSCs were transferred. Efficient engraftment of human CD45+ cells was also observed in non-irradiated NOGW mice transferred with 20,000-40,000 HSCs. The bone marrow (BM) of NOGW mice exhibited significantly more engrafted human HSCs or progenitor cells (CD34+CD38- or CD34+CD38+ cells) than the BM of NOG mice. Furthermore, we generated a human cytokine (interleukin-3 and granulocyte-macrophage colony-stimulating factor) transgenic NOG-W41 (NOGW-EXL) mouse to achieve multilineage reconstitution with sufficient engraftment of human hematopoietic cells. Non-irradiated NOGW-EXL mice showed significantly higher engraftment levels of human CD45+ and myeloid lineage cells, particularly granulocytes and platelets/megakaryocytes, than non-irradiated NOGW or irradiated NOG-EXL mice after human CD34+ cell transplantation. Serial BM transplantation experiments revealed that NOGW mice exhibited the highest potential for long-term HSC compared with other strains. Consequently, c-kit mutant NOGW-EXL humanized mice represent an advanced model for HSC-transferred humanized mice and hold promise for widespread applications owing to their high versatility.

Keywords:  CD34+ cells; Hematopoiesis; Hematopoietic stem cells; Humanized mice; NOG

DOI:  https://doi.org/10.1186/s13287-024-03799-w
Cancer Lett. 2024 Jun 14. pii: S0304-3835(24)00454-3. [Epub ahead of print] 217060

HIF-2α Inhibition Disrupts Leukemia Stem Cell Metabolism and Impairs Vascular Microenvironment to Enhance Chronic Myeloid Leukemia Treatment.

Jian Wang, Weiwei Ma, Junbin Huang, Guo Qiu, Tian Zhang, Qinghua Wei, Chong He, Dunhua Zhou, Meng Zhao, Chun Chen, Xi Xu.

Leukemic stem cells (LSCs) in chronic myeloid leukemia (CML) contribute to treatment resistance and disease recurrence. Metabolism regulates LSCs, but the mechanisms remain elusive. Here, we show that hypoxia-inducible factor 2α (HIF-2α) is highly expressed in LSCs in mouse and human CML and increases after tyrosine kinase inhibitor (TKI) treatment. Deletion of HIF-2α suppresses disease progression, reduces LSC numbers, and enhances the efficacy of TKI treatment in BCL-ABL-induced CML mice. Mechanistically, HIF-2α deletion reshapes the metabolic profile of LSCs, leading to increased production of reactive oxygen species (ROS) and apoptosis in CML. Moreover, HIF-2α deletion decreases vascular endothelial growth factor (VEGF) expression, thereby suppressing neovascularization in the bone marrow of CML mice. Furthermore, pharmaceutical inhibition of HIF-2α by PT2399 attenuates disease progression and improves the efficacy of TKI treatment in both mouse and human CML. Overall, our findings highlight the role of HIF-2α in controlling the metabolic state and vascular niche remodeling in CML, suggesting it is a potential therapeutic target to enhance TKI therapy.

DOI: https://doi.org/10.1016/j.canlet.2024.217060
bioRxiv. 2024 Jun 04. pii: 2024.06.04.597454. [Epub ahead of print]

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

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

Dysregulated intracellular pH (pHi) dynamics and an altered tumor microenvironment have emerged as drivers of cancer cell phenotypes. However, the molecular integration between the physical properties of the microenvironment and dynamic intracellular signaling responses remains unclear. Here, we use two metastatic cell models, one breast and one lung, to assess pHi response to varying extracellular matrix (ECM) stiffness. To experimentally model ECM stiffening, we use two tunable-stiffness hydrogel systems: Matrigel and hyaluronic acid (HA) gels, which mimic the increased protein secretion and crosslinking associated with ECM stiffening. We find that single-cell pHi decreases with increased ECM stiffness in both hydrogel systems and both metastatic cell types. We also observed that stiff ECM promotes vasculogenic mimicry (VM), a phenotype associated with metastasis and resistance. Importantly, we show that decreased pHi is both a necessary and sufficient mediator of VM, as raising pHi on stiff ECM reduces VM phenotypes and lowering pHi on soft ECM drives VM. We characterize β-catenin as a pH-dependent molecular mediator of pH-dependent VM, where stiffness-driven changes in β-catenin abundance can be overridden by increased pHi. We uncover a dynamic relationship between matrix stiffness and pHi, thus suggesting pHi dynamics can override mechanosensitive cell responses to the extracellular microenvironment.

DOI: https://doi.org/10.1101/2024.06.04.597454
bioRxiv. 2024 Jun 08. pii: 2024.06.07.596542. [Epub ahead of print]

Hematopoietic stem cell division is governed by distinct RUNX1 binding partners.

Anne L Robertson, Lu Yue, Avik Choudhuri, Caroline Kubaczka, Samuel J Wattrus, Joseph Mandelbaum, Serine Avagyan, Song Yang, Rebecca J Freeman, Victoria Chan, Megan C Blair, George Q Daley, Leonard I Zon.

A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.

DOI: https://doi.org/10.1101/2024.06.07.596542
Int J Mol Sci. 2024 May 30. pii: 6020. [Epub ahead of print]25(11):

Distinct Responses to Menin Inhibition and Synergy with DOT1L Inhibition in KMT2A-Rearranged Acute Lymphoblastic and Myeloid Leukemia.

Fabienne R S Adriaanse, Pauline Schneider, Susan T C J M Arentsen-Peters, Ana M Neves da Fonseca, Janine Stutterheim, Rob Pieters, C Michel Zwaan, Ronald W Stam.

  Pediatric acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) exhibit favorable survival rates. However, for AML and ALL patients carrying KMT2A gene translocations clinical outcome remains unsatisfactory. Key players in KMT2A-fusion-driven leukemogenesis include menin and DOT1L. Recently, menin inhibitors like revumenib have garnered attention for their potential therapeutic efficacy in treating KMT2A-rearranged acute leukemias. However, resistance to menin inhibition poses challenges, and identifying which patients would benefit from revumenib treatment is crucial. Here, we investigated the in vitro response to revumenib in KMT2A-rearranged ALL and AML. While ALL samples show rapid, dose-dependent induction of leukemic cell death, AML responses are much slower and promote myeloid differentiation. Furthermore, we reveal that acquired resistance to revumenib in KMT2A-rearranged ALL cells can occur either through the acquisition of MEN1 mutations or independently of mutations in MEN1. Finally, we demonstrate significant synergy between revumenib and the DOT1L inhibitor pinometostat in KMT2A-rearranged ALL, suggesting that such drug combinations represent a potent therapeutic strategy for these patients. Collectively, our findings underscore the complexity of resistance mechanisms and advocate for precise patient stratification to optimize the use of menin inhibitors in KMT2A-rearranged acute leukemia.

Keywords:  ALL; AML; DOT1L; KMT2A-rearranged; infant; menin; pediatric; pinometostat; revumenib; synergy

DOI:  https://doi.org/10.3390/ijms25116020
Int J Biol Sci. 2024 ;20(8): 3140-3155

CYR61 Acts as an Intracellular Microtubule-Associated Protein and Coordinates Mitotic Progression via PLK1-FBW7 Pathway.

Kaishun Hu, Limin Xie, Wenjing Wu, Jingyuan Zhang, Yu Li, Jiehua He, Yin Zhang, Daning Lu, H Phillip Koeffler, Lehang Lin, Dong Yin.

  Cysteine-rich angiogenic inducer 61 (CYR61), also called CCN1, has long been characterized as a secretory protein. Nevertheless, the intracellular function of CYR61 remains unclear. Here, we found that CYR61 is important for proper cell cycle progression. Specifically, CYR61 interacts with microtubules and promotes microtubule polymerization to ensure mitotic entry. Moreover, CYR61 interacts with PLK1 and accumulates during the mitotic process, followed by degradation as mitosis concludes. The proteolysis of CYR61 requires the PLK1 kinase activity, which directly phosphorylates two conserved motifs on CYR61, enhancing its interaction with the SCF E3 complex subunit FBW7 and mediating its degradation by the proteasome. Mutations of phosphorylation sites of Ser167 and Ser188 greatly increase CYR61's stability, while deletion of CYR61 extends prophase and metaphase and delays anaphase onset. In summary, our findings highlight the precise control of the intracellular CYR61 by the PLK1-FBW7 pathway, accentuating its significance as a microtubule-associated protein during mitotic progression.

Keywords:  CYR61; FBW7; PLK1; microtubules; mitosis

DOI:  https://doi.org/10.7150/ijbs.93335
Br J Haematol. 2024 Jun 18.

GATA2 heterozygosity causes an epigenetic feedback mechanism resulting in myeloid and erythroid dysplasia.

Emanuele Gioacchino, Wei Zhang, Cansu Koyunlar, Joke Zink, Hans de Looper, Kirsten J Gussinklo, Remco Hoogenboezem, Dennis Bosch, Eric Bindels, Ivo P Touw, Emma de Pater.

  The transcription factor GATA2 has a pivotal role in haematopoiesis. Heterozygous germline GATA2 mutations result in a syndrome characterized by immunodeficiency, bone marrow failure and predispositions to myelodysplastic syndrome (MDS) and acute myeloid leukaemia. Clinical symptoms in these patients are diverse and mechanisms driving GATA2-related phenotypes are largely unknown. To explore the impact of GATA2 haploinsufficiency on haematopoiesis, we generated a zebrafish model carrying a heterozygous mutation of gata2b (gata2b+/-), an orthologue of GATA2. Morphological analysis revealed myeloid and erythroid dysplasia in gata2b+/- kidney marrow. Because Gata2b could affect both transcription and chromatin accessibility during lineage differentiation, this was assessed by single-cell (sc) RNA-seq and single-nucleus (sn) ATAC-seq. Sn-ATAC-seq showed that the co-accessibility between the transcription start site (TSS) and a -3.5-4.1 kb putative enhancer was more robust in gata2b+/- zebrafish HSPCs compared to wild type, increasing gata2b expression and resulting in higher genome-wide Gata2b motif use in HSPCs. As a result of increased accessibility of the gata2b locus, gata2b+/- chromatin was also more accessible during lineage differentiation. scRNA-seq data revealed myeloid differentiation defects, that is, impaired cell cycle progression, reduced expression of cebpa and cebpb and increased signatures of ribosome biogenesis. These data also revealed a differentiation delay in erythroid progenitors, aberrant proliferative signatures and down-regulation of Gata1a, a master regulator of erythropoiesis, which worsened with age. These findings suggest that cell-intrinsic compensatory mechanisms, needed to obtain normal levels of Gata2b in heterozygous HSPCs to maintain their integrity, result in aberrant lineage differentiation, thereby representing a critical step in the predisposition to MDS.

Keywords:  GATA2 deficiency syndrome; Gata2b; HSCs; MDS; epigenetic compensation; zebrafish

DOI:  https://doi.org/10.1111/bjh.19585
Proc Natl Acad Sci U S A. 2024 Jun 25. 121(26): e2317945121

Calorie restriction and calorie-restriction mimetics activate chaperone-mediated autophagy.

Maryam Jafari, Adrián Macho-González, Antonio Diaz, Kristen Lindenau, Olaya Santiago-Fernández, Mei Zeng, Ashish C Massey, Rafael de Cabo, Susmita Kaushik, Ana Maria Cuervo.

  Chaperone-mediated autophagy (CMA) is part of the mammalian cellular proteostasis network that ensures protein quality control, maintenance of proteome homeostasis, and proteome changes required for the adaptation to stress. Loss of proteostasis is one of the hallmarks of aging. CMA decreases with age in multiple rodent tissues and human cell types. A decrease in lysosomal levels of the lysosome-associated membrane protein type 2A (LAMP2A), the CMA receptor, has been identified as a main reason for declined CMA in aging. Here, we report constitutive activation of CMA with calorie restriction (CR), an intervention that extends healthspan, in old rodent livers and in an in vitro model of CR with cultured fibroblasts. We found that CR-mediated upregulation of CMA is due to improved stability of LAMP2A at the lysosome membrane. We also explore the translational value of our observations using calorie-restriction mimetics (CRMs), pharmacologically active substances that reproduce the biochemical and functional effects of CR. We show that acute treatment of old mice with CRMs also robustly activates CMA in several tissues and that this activation is required for the higher resistance to lipid dietary challenges conferred by treatment with CRMs. We conclude that part of the beneficial effects associated with CR/CRMs could be a consequence of the constitutive activation of CMA mediated by these interventions.

Keywords:  aging; autophagy; dietary restriction; gerotherapeutics; lysosomes

DOI:  https://doi.org/10.1073/pnas.2317945121
Stem Cell Res Ther. 2024 Jun 18. 15(1): 178

Extracellular matrices of stromal cell subtypes regulate phenotype and contribute to the stromal microenvironment in vivo.

Andrew P Stone, Emma Rand, Gabriel Thornes, Alasdair G Kay, Amanda L Barnes, Ian S Hitchcock, Paul G Genever.

   BACKGROUND: Bone marrow stromal cells (BMSCs) are highly heterogeneous, which may reflect their diverse biological functions, including tissue maintenance, haematopoietic support and immune control. The current understanding of the mechanisms that drive the onset and resolution of heterogeneity, and how BMSCs influence other cells in their environment is limited. Here, we determined how the secretome and importantly the extracellular matrix of BMSCs can influence cellular phenotype.
METHODS: We used two immortalised clonal BMSC lines isolated from the same heterogeneous culture as model stromal subtypes with distinct phenotypic traits; a multipotent stem-cell-like stromal line (Y201) and a nullipotent non-stem cell stromal line (Y202), isolated from the same donor BMSC pool. Label-free quantitative phase imaging was used to track cell morphology and migration of the BMSC lines over 96 h in colony-forming assays. We quantified the secreted factors of each cell line by mass spectrometry and confirmed presence of proteins in human bone marrow by immunofluorescence.
RESULTS: Transfer of secreted signals from a stem cell to a non-stem cell resulted in a change in morphology and enhanced migration to more closely match stem cell-like features. Mass spectrometry analysis revealed a significant enrichment of extracellular matrix (ECM) proteins in the Y201 stem cell secretome compared to Y202 stromal cells. We confirmed that Y201 produced a more robust ECM in culture compared to Y202. Growth of Y202 on ECM produced by Y201 or Y202 restored migration and fibroblastic morphology, suggesting that it is the deficiency of ECM production that contributes to its phenotype. The proteins periostin and aggrecan, were detected at 71- and 104-fold higher levels in the Y201 versus Y202 secretome and were subsequently identified by immunofluorescence at rare sites on the endosteal surfaces of mouse and human bone, underlying CD271-positive stromal cells. These proteins may represent key non-cellular components of the microenvironment for bona-fide stem cells important for cell maintenance and phenotype in vivo.
CONCLUSIONS: We identified plasticity in BMSC morphology and migratory characteristics that can be modified through secreted proteins, particularly from multipotent stem cells. Overall, we demonstrate the importance of specific ECM proteins in co-ordination of cellular phenotype and highlight how non-cellular components of the BMSC microenvironment may provide insights into cell population heterogeneity and the role of BMSCs in health and disease.

Keywords:  Bone marrow stromal cell; Cellular heterogeneity; Extracellular matrix; Secretome; Stromal microenvironment

DOI:  https://doi.org/10.1186/s13287-024-03786-1
Nat Commun. 2024 Jun 17. 15(1): 5152

C-terminally phosphorylated p27 activates self-renewal driver genes to program cancer stem cell expansion, mammary hyperplasia and cancer.

Seyedeh Fatemeh Razavipour, Hyunho Yoon, Kibeom Jang, Minsoon Kim, Hend M Nawara, Amir Bagheri, Wei-Chi Huang, Miyoung Shin, Dekuang Zhao, Zhiqun Zhou, Derek Van Boven, Karoline Briegel, Lluis Morey, Tan A Ince, Michael Johnson, Joyce M Slingerland.

In many cancers, a stem-like cell subpopulation mediates tumor initiation, dissemination and drug resistance. Here, we report that cancer stem cell (CSC) abundance is transcriptionally regulated by C-terminally phosphorylated p27 (p27pT157pT198). Mechanistically, this arises through p27 co-recruitment with STAT3/CBP to gene regulators of CSC self-renewal including MYC, the Notch ligand JAG1, and ANGPTL4. p27pTpT/STAT3 also recruits a SIN3A/HDAC1 complex to co-repress the Pyk2 inhibitor, PTPN12. Pyk2, in turn, activates STAT3, creating a feed-forward loop increasing stem-like properties in vitro and tumor-initiating stem cells in vivo. The p27-activated gene profile is over-represented in STAT3 activated human breast cancers. Furthermore, mammary transgenic expression of phosphomimetic, cyclin-CDK-binding defective p27 (p27CK-DD) increases mammary duct branching morphogenesis, yielding hyperplasia and microinvasive cancers that can metastasize to liver, further supporting a role for p27pTpT in CSC expansion. Thus, p27pTpT interacts with STAT3, driving transcriptional programs governing stem cell expansion or maintenance in normal and cancer tissues.

DOI: https://doi.org/10.1038/s41467-024-48742-y
Biochemistry (Mosc). 2024 May;89(5): 883-903

Molecular Changes in Immunological Characteristics of Bone Marrow Multipotent Mesenchymal Stromal Cells in Lymphoid Neoplasia.

Nataliya A Petinati, Aleksandra V Sadovskaya, Natalia V Sats, Nikolai M Kapranov, Yulia O Davydova, Ekaterina A Fastova, Aminat U Magomedova, Anastasia N Vasilyeva, Olga A Aleshina, Georgiy P Arapidi, Viktoria O Shender, Igor P Smirnov, Olga V Pobeguts, Maria A Lagarkova, Nina I Drize, Elena N Parovichnikova.

  Immune system and bone marrow stromal cells play an important role in maintaining normal hematopoiesis. Lymphoid neoplasia disturbs not only development of immune cells, but other immune response mechanisms as well. Multipotent mesenchymal stromal cells (MSCs) of the bone marrow are involved in immune response regulation through both intercellular interactions and secretion of various cytokines. In hematological malignancies, the bone marrow stromal microenvironment, including MSCs, is altered. Aim of this study was to describe the differences of MSCs' immunological function in the patients with acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). In ALL, malignant cells arise from the early precursor cells localized in bone marrow, while in DLBCL they arise from more differentiated B-cells. In this study, only the DLBCL patients without bone marrow involvement were included. Growth parameters, surface marker expression, genes of interest expression, and secretion pattern of bone marrow MSCs from the patients with ALL and DLBCL at the onset of the disease and in remission were studied. MSCs from the healthy donors of corresponding ages were used as controls. It has been shown that concentration of MSCs in the bone marrow of the patients with ALL is reduced at the onset of the disease and is restored upon reaching remission; in the patients with DLBCL this parameter does not change. Proliferative capacity of MSCs did not change in the patients with ALL; however, the cells of the DLBCL patients both at the onset and in remission proliferated significantly faster than those from the donors. Expression of the membrane surface markers and expression of the genes important for differentiation, immunological status maintenance, and cytokine secretion differed significantly in the MSCs of the patients from those of the healthy donors and depended on nosology of the disease. Secretomes of the MSCs varied greatly; a number of proteins associated with immune response regulation, differentiation, and maintenance of hematopoietic stem cells were depleted in the secretomes of the cells from the patients. Lymphoid neoplasia leads to dramatic changes in the functional immunological status of MSCs.

Keywords:  acute lymphoblastic leukemia; diffuse large B-cell lymphoma; gene expression; multipotent mesenchymal stromal cells; protein secretion

DOI:  https://doi.org/10.1134/S0006297924050092
Biochem Biophys Rep. 2024 Sep;39 101737

Step by step analysis on gene datasets of growth phases in hematopoietic stem cells.

Mohammad Elahimanesh, Nafiseh Shokri, Payam Mohammadi, Najmeh Parvaz, Mohammad Najafi.

   Background: Umbilical cord blood hematopoietic stem cells (UCB-HSCs) have important roles in the treatment of illnesses based on their self-renewal and potency characteristics. Knowing the gene profiles and signaling pathways involved in each step of the cell cycle could improve the therapeutic approaches of HSCs. The aim of this study was to predict the gene profiles and signaling pathways involved in the G0, G1, and differentiation stages of HSCs.
Methods: Interventional (n = 8) and non-interventional (n = 3) datasets were obtained from the Gene Expression Omnibus (GEO) database, and were crossed and analyzed to determine the high- and low-express genes related to each of the G0, G1, and differentiation stages of HSCs. Then, the scores of STRING were annotated to the gene data. The gene networks were constructed using Cytoscape software, and enriched with the KEGG and GO databases.
Results: The high- and low-express genes were determined due to inter and intra intersections of the interventional and non-interventional data. The non-interventional data were applied to construct the gene networks (n = 6) with the nodes improved using the interventional data. Several important signaling pathways were suggested in each of the G0, G1, and differentiation stages.
Conclusion: The data revealed that the different signaling pathways are activated in each of the G0, G1, and differentiation stages so that their genes may be targeted to improve the HSC therapy.

Keywords:  Gene ontology; HSC phases; Hematopoietic stem cell; Microarray; Signaling pathways

DOI:  https://doi.org/10.1016/j.bbrep.2024.101737
Commun Biol. 2024 Jun 20. 7(1): 753

Protein arginine methyltransferase 2 controls inflammatory signaling in acute myeloid leukemia.

Camille Sauter, Thomas Morin, Fabien Guidez, John Simonet, Cyril Fournier, Céline Row, Denis Masnikov, Baptiste Pernon, Anne Largeot, Aziza Aznague, Yann Hérault, Guy Sauvageau, Marc Maynadié, Mary Callanan, Jean-Noël Bastie, Romain Aucagne, Laurent Delva.

Arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and is involved in various cellular processes, including cancer development. PRMT2 expression is increased in several cancer types although its role in acute myeloid leukemia (AML) remains unknown. Here, we investigate the role of PRMT2 in a cohort of patients with AML, PRMT2 knockout AML cell lines as well as a Prmt2 knockout mouse model. In patients, low PRMT2 expressors are enriched for inflammatory signatures, including the NF-κB pathway, and show inferior survival. In keeping with a role for PRMT2 in control of inflammatory signaling, bone marrow-derived macrophages from Prmt2 KO mice display increased pro-inflammatory cytokine signaling upon LPS treatment. In PRMT2-depleted AML cell lines, aberrant inflammatory signaling has been linked to overproduction of IL6, resulting from a deregulation of the NF-κB signaling pathway, therefore leading to hyperactivation of STAT3. Together, these findings identify PRMT2 as a key regulator of inflammation in AML.

DOI: https://doi.org/10.1038/s42003-024-06453-6
Elife. 2024 Jun 18. pii: RP91611. [Epub ahead of print]12

SOD1 is a synthetic-lethal target in PPM1D-mutant leukemia cells.

Linda Zhang, Joanne I Hsu, Etienne D Braekeleer, Chun-Wei Chen, Tajhal D Patel, Alejandra G Martell, Anna G Guzman, Katharina Wohlan, Sarah M Waldvogel, Hidetaka Uryu, Ayala Tovy, Elsa Callen, Rebecca L Murdaugh, Rosemary Richard, Sandra Jansen, Lisenka Vissers, Bert B A de Vries, Andre Nussenzweig, Shixia Huang, Cristian Coarfa, Jamie Anastas, Koichi Takahashi, George Vassiliou, Margaret A Goodell.

  The DNA damage response is critical for maintaining genome integrity and is commonly disrupted in the development of cancer. PPM1D (protein phosphatase Mg2+/Mn2+-dependent 1D) is a master negative regulator of the response; gain-of-function mutations and amplifications of PPM1D are found across several human cancers making it a relevant pharmacological target. Here, we used CRISPR/Cas9 screening to identify synthetic-lethal dependencies of PPM1D, uncovering superoxide dismutase-1 (SOD1) as a potential target for PPM1D-mutant cells. We revealed a dysregulated redox landscape characterized by elevated levels of reactive oxygen species and a compromised response to oxidative stress in PPM1D-mutant cells. Altogether, our results demonstrate a role for SOD1 in the survival of PPM1D-mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D-mutant cancers.

Keywords:  DNA damage; cancer; cancer biology; cell biology; leukemia; mouse

DOI:  https://doi.org/10.7554/eLife.91611
Sci Signal. 2024 Jun 18. 17(841): eadi8743

Pin1-catalyzed conformational regulation after phosphorylation: A distinct checkpoint in cell signaling and drug discovery.

Kun Ping Lu, Xiao Zhen Zhou.

Protein phosphorylation is one of the most common mechanisms regulating cellular signaling pathways, and many kinases and phosphatases are proven drug targets. Upon phosphorylation, protein functions can be further regulated by the distinct isomerase Pin1 through cis-trans isomerization. Numerous protein targets and many important roles have now been elucidated for Pin1. However, no tools are available to detect or target cis and trans conformation events in cells. The development of Pin1 inhibitors and stereo- and phospho-specific antibodies has revealed that cis and trans conformations have distinct and often opposing cellular functions. Aberrant conformational changes due to the dysregulation of Pin1 can drive pathogenesis but can be effectively targeted in age-related diseases, including cancers and neurodegenerative disorders. Here, we review advances in understanding the roles of Pin1 signaling in health and disease and highlight conformational regulation as a distinct signal transduction checkpoint in disease development and treatment.

DOI: https://doi.org/10.1126/scisignal.adi8743
bioRxiv. 2024 May 24. pii: 2024.05.23.595547. [Epub ahead of print]

Single-cell proteomics reveals decreased abundance of proteostasis and meiosis proteins in advanced maternal age oocytes.

S Galatidou, A Petelski, A Pujol, K Lattes, L B Latorraca, T Fair, M Popovic, R Vassena, N Slavov, M Barragan.

Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this age-related decline remain unclear. To gain insights into this phenomenon, we applied plexDIA, a multiplexed, single-cell mass spectrometry method, to analyze the proteome of oocytes from both young women and women of advanced maternal age. Our findings primarily revealed distinct proteomic profiles between immature fully grown germinal vesicle and mature metaphase II oocytes. Importantly, we further show that a woman's age is associated with changes in her oocyte proteome. Specifically, when compared to oocytes obtained from young women, advanced maternal age oocytes exhibited lower levels of the proteasome and TRiC complex, as well as other key regulators of proteostasis and meiosis. This suggests that aging adversely affects the proteostasis and meiosis networks in human oocytes. The proteins identified in this study hold potential as targets for improving oocyte quality and may guide future studies into the molecular processes underlying oocyte aging.

DOI: https://doi.org/10.1101/2024.05.23.595547
bioRxiv. 2024 Mar 13. pii: 2024.03.11.584337. [Epub ahead of print]

Astrocytes control quiescent NSC reactivation via GPCR signaling-mediated F-actin remodeling.

Kun-Yang Lin, Mahekta R Gujar, Jiaen Lin, Wei Yung Ding, Jiawen Huang, Yang Gao, Ye Sing Tan, Xiang Teng, Low Siok Lan Christine, Pakorn Kanchanawong, Yusuke Toyama, Hongyan Wang.

The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. Drosophila quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. Here, we reveal the fine F-actin structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. We show that F-actin polymerization promotes the nuclear translocation of Mrtf, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G-protein-coupled receptor (GPCR) Smog, G-protein αq subunit, Rho1 GTPase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand Fog to regulate Gαq-Rho1-Dia-mediated NSC reactivation. Together, we establish that the Smog-Gαq-Rho1 signaling axis derived from astrocytes, a NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.

DOI: https://doi.org/10.1101/2024.03.11.584337
Br J Haematol. 2024 Jun 15.

Synergistic effects of the KDM4C inhibitor SD70 and the menin inhibitor MI-503 against MLL::AF9-driven acute myeloid leukaemia.

Wenqi Zhu, Yiyi Ding, Wanling Huang, Nini Guo, Qian Ren, Nan Wang, Xiaotong Ma.

  MLL-rearranged (MLL-r) leukaemia is observed in approximately 10% of acute myeloid leukaemia (AML) and is associated with a relatively poor prognosis, highlighting the need for new treatment regimens. MLL fusion proteins produced by MLL rearrangements recruit KDM4C to mediate epigenetic reprogramming, which is required for the maintenance of MLL-r leukaemia. In this study, we used a combinatorial drug screen to selectively identify synergistic treatment partners for the KDM4C inhibitor SD70. The results showed that the drug combination of SD70 and MI-503, a potent menin-MLL inhibitor, induced synergistically enhanced apoptosis in MLL::AF9 leukaemia cells without affecting normal CD34+ cells. In vivo treatment with SD70 and MI-503 significantly prolonged survival in AML xenograft models. Differential gene expression analysis by RNA-seq following combined pharmacological inhibition of SD70 and MI-503 revealed changes in numerous genes, with MYC target genes being the most significantly downregulated. Taken together, these data provide preclinical evidence that the combination of SD70 and MI-503 is a potential dual-targeted therapy for MLL::AF9 AML.

Keywords:   MLL::AF9 ; AML; MI‐503; SD70; combination therapy

DOI:  https://doi.org/10.1111/bjh.19591
Pharmacol Ther. 2024 Jun 13. pii: S0163-7258(24)00100-1. [Epub ahead of print]260 108680

UFMylation: An integral post-translational modification for the regulation of proteostasis and cellular functions.

Xiaohui Wang, Xiaowei Lv, Jingjing Ma, Guoqiang Xu.

  Ubiquitin-fold modifier 1 (UFM1) is covalently conjugated to protein substrates via a cascade of enzymatic reactions, a process known as UFMylation. UFMylation orchestrates an array of vital biological functions, including maintaining endoplasmic reticulum (ER) homeostasis, facilitating protein biogenesis, promoting cellular differentiation, regulating DNA damage response, and participating in cancer-associated signaling pathways. UFMylation has rapidly evolved into one of the forefront research areas within the last few years, yet much remains to be uncovered. In this review, first, UFMylation and its cellular functions associated with diseases are briefly introduced. Then, we summarize the proteomic approaches for identifying UFMylation substrates and explore the impact of UFMylation on gene transcription, protein translation, and maintenance of ER homeostasis. Next, we highlight the intricate regulation between UFMylation and two protein degradation pathways, the ubiquitin-proteasome system and the autophagy-lysosome pathway, and explore the potential of UFMylation system as a drug target. Finally, we discuss emerging perspectives in the UFMylation field. This review may provide valuable insights for drug discovery targeting the UFMylation system.

Keywords:  ALP; ER homeostasis; Gene transcription; Protein translation; UFMylation; UPS

DOI:  https://doi.org/10.1016/j.pharmthera.2024.108680
Methods Mol Biol. 2024 ;2817 157-175

Single-Cell Proteomics Analysis with Tecan Uno and SCREEN Workflow.

Michael Lewandowski, Shad Morton, Matthew Blake, Erica Squires, Rushdy Ahmad, David R Walt, Bogdan Budnik.

  With advances in sample preparation, small-volume liquid dispensing technologies, high-resolution MS/MS instrumentation, and data acquisition methodologies, it has become increasingly possible to confidently investigate the heterogeneous proteome found within individual cells. In this chapter, we present an automated high-throughput sample preparation workflow based on the Tecan Uno instrument for quantitative single-cell mass spectrometry-based proteomics. Cells are analyzed by the Single-Cell Proteome Analysis platform (SCREEN), which was introduced earlier and provides deeper proteome coverage across single cells.

Keywords:  Automation; High-throughput sample preparation; SCREEN method; Tecan Uno

DOI:  https://doi.org/10.1007/978-1-0716-3934-4_13
Blood. 2024 Jun 20. 143(25): 2675

Azab AK, Quang P, Azab F, et al. P-selectin glycoprotein ligand regulates the interaction of multiple myeloma cells with the bone marrow microenvironment. Blood. 2012;119(6):1468-1478.

DOI: https://doi.org/10.1182/blood.2024024988
Methods Mol Biol. 2024 ;2817 1-7

Label-Free Sample Preparation for Single-Cell Proteomics.

David Hartlmayr, Claudia Ctortecka, Rupert Mayer, Karl Mechtler, Anjali Seth.

  In recent years, single-cell proteomics (SCP) has become a valuable addition to other single-cell omics technologies for studying cellular heterogeneity. The amount of protein in a single cell is very limited, and in contrast to sequencing techniques, there are currently no means for protein amplification. Therefore, most single-cell proteomics approaches aim to maximize sample preparation efficiency while minimizing peptide loss. By reducing processing volumes to sub-microliters and avoiding manual transfer steps that could lead to peptide loss, peptide recovery, and the robustness of SCP workflows have been significantly improved. In this chapter, we describe a protocol for label-free SCP sample preparation using the cellenONE® platform and the proteoCHIP LF 48 substrate prior to analysis with high-performance liquid chromatography-mass spectrometry.

Keywords:  Automation; Label-free; Single-cell proteomics; Sub-μL sample preparation; cellenONE®; proteoCHIP LF 48

DOI:  https://doi.org/10.1007/978-1-0716-3934-4_1
Methods Mol Biol. 2024 ;2817 97-113

Spatial Proteomics of Single Hepatocytes with Multiplexed Data-Independent Acquisition (mDIA).

Marvin Thielert, Caroline A M Weiss, Matthias Mann, Florian A Rosenberger.

  Spatially resolved mass spectrometry-based proteomics at single-cell resolution promises to provide insights into biological heterogeneity. We describe a protocol based on multiplexed data-independent acquisition (mDIA) with dimethyl labeling to enhance proteome depth, accuracy, and throughput while minimizing costs. It enables high-quality proteome analysis of single isolated hepatocytes and utilizes liver zonation for single-cell proteomics benchmarking. This adaptable, modular protocol will promote the use of single-cell proteomics in spatial biology.

Keywords:  DIA; Hepatocyte; Liver; Mass spectrometry; Multiplexing; Single-cell proteomics; Spatial proteomics

DOI:  https://doi.org/10.1007/978-1-0716-3934-4_9
Cancer Res. 2024 Jun 17.

Oncogenic Calreticulin Induces Immune Escape by Stimulating TGF-β Expression and Regulatory T Cell Expansion in the Bone Marrow Microenvironment.

Dominik Schmidt, Cornelia Endres, Rouven Hoefflin, Geoffroy Andrieux, Melissa Zwick, Nikolaos Karantzelis, Hans F Staehle, Janaki Manoja Vinnakota, Sandra Duquesne, Miriam Mozaffari Jovein, Dietmar Pfeifer, Heiko Becker, Bruce R Blazar, Alexander Zähringer, Justus Duyster, Tilman Brummer, Melanie Boerries, Julian Baumeister, Khalid Shoumariyeh, Juan Li, Anthony R Green, Florian H Heidel, Itay Tirosh, Heike L Pahl, Nils Leimkühler, Natalie Köhler, Marcelo A S de Toledo, Steffen Koschmieder, Robert Zeiser.

Increasing evidence supports the interplay between oncogenic mutations and immune escape mechanisms. Strategies to counteract the immune escape mediated by oncogenic signaling could provide improved therapeutic options for patients with various malignancies. As mutant calreticulin (CALR) is a common driver of myeloproliferative neoplasms (MPN), we analyzed the impact of oncogenic CALRdel52 on the bone marrow (BM) microenvironment in MPN. Single-cell RNA-sequencing revealed that CALRdel52 led to the expansion of TGF-β1-producing erythroid progenitor cells and promoted the expansion of FoxP3+ regulatory T cells (Treg) in a murine MPN model. Treatment with an anti-TGF-β antibody improved mouse survival and increased the glycolytic activity in CD4+ and CD8+ T cells in vivo, while T cell depletion abrogated the protective effects conferred by neutralizing TGF-β. TGF-β1 reduced perforin and TNF-α production by T cells in vitro. TGF-β1 production by CALRdel52 cells was dependent on JAK1/2, PI3K, and ERK activity, which activated the transcription factor Sp1 to induce TGF-β1 expression. In four independent patient cohorts, TGF-β1 expression was increased in the BM of MPN patients compared to healthy individuals, and the BM of MPN patients contained a higher frequency of Treg compared to healthy individuals. Together, this study identified an ERK/Sp1/TGF-β1 axis in CALRdel52 MPNs as a mechanism of immunosuppression that can be targeted to elicit T-cell-mediated cytotoxicity.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-3553
Elife. 2024 Jun 17. pii: RP91999. [Epub ahead of print]12

Expression of modified FcγRI enables myeloid cells to elicit robust tumor-specific cytotoxicity.

Leen Farhat-Younis, Manho Na, Amichai Zarfin, Aseel Khateeb, Nadine Santana-Magal, Alon Richter, Amit Gutwillig, Diana Rasoulouniriana, Annette Gleiberman, Lir Beck, Tamar Giger, Avraham Ashkenazi, Adi Barzel, Peleg Rider, Yaron Carmi.

  Despite the central role of T cells in tumor immunity, attempts to harness their cytotoxic capacity as a therapy have met limited efficacy, partially as a result of the suppressive microenvironment which limits their migration and activation. In contrast, myeloid cells massively infiltrate tumors and are well adapted to survive these harsh conditions. While they are equipped with cell-killing abilities, they often adopt an immunosuppressive phenotype upon migration to tumors. Therefore, the questions of how to modify their activation programming against cancer, and what signaling cascades should be activated in myeloid cells to elicit their cytotoxicity have remained unclear. Here, we found that activation of IgM-induced signaling in murine myeloid cells results in secretion of lytic granules and massive tumor cell death. These findings open venues for designing novel immunotherapy by equipping monocytes with chimeric receptors that target tumor antigens and consequently, signal through IgM receptor. Nonetheless, we found that myeloid cells do not express the antibody-derived portion used to recognize the tumor antigen due to the induction of an ER stress response. To overcome this limitation, we designed chimeric receptors that are based on the high-affinity FcγRI for IgG. Incubation of macrophages expressing these receptors along with tumor-binding IgG induced massive tumor cell killing and secretion of reactive oxygen species and Granzyme B. Overall, this work highlights the challenges involved in genetically reprogramming the signaling in myeloid cells and provides a framework for endowing myeloid cells with antigen-specific cytotoxicity.

Keywords:  ER stress; chimeric antigen receptor; immunology; immunotherapy; inflammation; mouse; myeloid cells

DOI:  https://doi.org/10.7554/eLife.91999
Blood Cancer Discov. 2024 Jun 21. OF1-OF15

Recent Advances in Immune-Based Therapies for Acute Myeloid Leukemia.

Cecilia Restelli, Marco Ruella, Luca Paruzzo, Corrado Tarella, Pier Giuseppe Pelicci, Emanuela Colombo.

Despite advancements, acute myeloid leukemia (AML) remains unconquered by current therapies. Evidence of immune evasion during AML progression, such as HLA loss and T-cell exhaustion, suggests that antileukemic immune responses contribute to disease control and could be harnessed by immunotherapy. In this review, we discuss a spectrum of AML immunotherapy targets, encompassing cancer cell-intrinsic and surface antigens as well as targeting in the leukemic milieu, and how they can be tailored for personalized approaches. These targets are overviewed across major immunotherapy modalities applied to AML: immune checkpoint inhibitors, antibody-drug conjugates, therapeutic vaccines, bispecific/trispecific antibodies, and chimeric antigen receptor (CAR)-T and CAR-NK cells. Significance: Immune therapies in AML treatment show evolving promise. Ongoing research aims to customize approaches for varied patient profiles and clinical scenarios. This review covers immune surveillance mechanisms, therapy options like checkpoint inhibitors, antibodies, CAR-T/NK cells, and vaccines, as well as resistance mechanisms and microenvironment considerations.

DOI: https://doi.org/10.1158/2643-3230.BCD-23-0202