bims-scepro Biomed News
on Stem cell proteostasis
Issue of 2024–11–24
seventeen papers selected by
William Grey, University of York



  1. Hemasphere. 2024 Nov;8(11): e70055
      Acute myeloid leukemia (AML) derives from hematopoietic stem and progenitor cells (HSPCs). To date, no AML-exclusive, non-HSPC-expressed cell-surface target molecules for AML selective immunotherapy have been identified. Therefore, to still apply surface-directed immunotherapy in this disease setting, time-limited combined immune-targeting of AML cells and healthy HSPCs, followed by hematopoietic stem cell transplantation (HSCT), might be a viable therapeutic approach. To explore this, we generated a recombinant single-chain variable fragment-based bispecific T-cell engaging and activating antibody directed against CD3 on T-cells and CD117, the surface receptor for stem cell factor, expressed by both AML cells and healthy HSPCs. Bispecific CD117xCD3 targeting induced lysis of CD117-positive healthy human HSPCs, AML cell lines and patient-derived AML blasts in the presence of T-cells at subnanomolar concentrations in vitro. Furthermore, in immunocompromised mice, engrafted with human CD117-expressing leukemia cells and human T-cells, the bispecific molecule efficiently prevented leukemia growth in vivo. Additionally, in immunodeficient mice transplanted with healthy human HSPCs, the molecule decreased the number of CD117-positive cells in vivo. Therefore, bispecific CD117xCD3 targeting might be developed clinically in order to reduce CD117-expressing leukemia cells and HSPCs prior to HSCT.
    DOI:  https://doi.org/10.1002/hem3.70055
  2. Leukemia. 2024 Nov 19.
      Polycomb group (PcG) proteins play important roles in hematopoietic stem cell (HSC) self-renewal. Mel18 and Bmi1 are homologs of the PCGF subunit within the Polycomb repressive complex 1 (PRC1). Bmi1 (PCGF4) enhances HSC self-renewal and promotes terminal differentiation. However, the role of Mel18 (PCGF2) in hematopoiesis is not fully understood and how Mel18 regulates gene transcription in HSCs remains elusive. We found that acute deletion of Mel18 in the hematopoietic compartment significantly increased the frequency of functional HSCs in the bone marrow. Furthermore, we demonstrate that Mel18 inhibits HSC self-renewal and proliferation. RNA-seq studies revealed that HSC self-renewal and proliferation gene signatures are enriched in Mel18-/- hematopoietic stem and progenitors (HSPCs) compared to Mel18+/+ HSPCs. Notably, ATAC-seq revealed increased chromatin accessibility at genes important for HSC self-renewal, whereas CUT&RUN showed decreased enrichment of H2AK119ub1 at genes important for proliferation, leading to increased expression of both Hoxb4 and Cdk4 in Mel18-/- HSPCs. Thus, we demonstrate that Mel18 inhibits hematopoietic stem cell self-renewal through repressing the transcription of genes important for HSC self-renewal and proliferation.
    DOI:  https://doi.org/10.1038/s41375-024-02462-w
  3. bioRxiv. 2024 Nov 05. pii: 2024.11.04.621964. [Epub ahead of print]
      Distinct routes of cellular production from hematopoietic stem cells (HSCs) have defined our current view of hematopoiesis. Recently, we challenged classical views of platelet generation, demonstrating that megakaryocyte progenitors (MkPs), and ultimately platelets, can be specified via an alternate and additive route of HSC-direct specification specifically during aging. This "shortcut" pathway generates hyperactive platelets likely to contribute to age-related platelet-mediated morbidities. Here, we used single-cell RNA/CITEseq to demonstrate that these age-unique, non-canonical (nc)MkPs can be prospectively defined and experimentally isolated from wild type mice. Surprisingly, this revealed that a rare population of ncMkPs also exist in young mice. Young and aged ncMkPs are functionally distinct from their canonical (c)MkP counterparts, with aged ncMkPs paradoxically and uniquely exhibiting enhanced survival and platelet generation capacity. We further demonstrate that aged HSCs generate significantly more ncMkPs than their younger counterparts, yet this is accomplished without strict clonal restriction. Together, these findings reveal significant phenotypic, functional, and aging-dependent heterogeneity among the MkP pool and uncover unique features of megakaryopoiesis throughout life, potentially offering cellular and molecular targets for mitigation of age-related adverse thrombotic events.
    DOI:  https://doi.org/10.1101/2024.11.04.621964
  4. Leukemia. 2024 Nov 21.
      Oncogenic programs regulate the proliferation and maintenance of cancer stem cells, and can define pharmacologic dependencies. In acute myeloid leukemia (AML) with the chromosome inversion 16 (inv(16)), the fusion oncoprotein CBFβ::MYH11 regulates pathways associated with leukemia stem cell activity. Here we demonstrate that expression of Neuropilin-1 (NRP1) is regulated by the fusion oncoprotein, and promotes AML expansion. Mechanistically, we show that the NRP1 locus has open chromatin in inv(16) AML, and that CBFβ::MYH11 modulates the local function of the transcription factors ERG, GATA2 and RUNX1 to sustain NRP1 levels. We found that ERG activates NRP1 expression, and that CBFβ::MYH11 knockdown represses ERG expression, thereby allowing the repressive activity of GATA2/RUNX1 at three NRP1 enhancers. Functionally, we demonstrate that NRP1 enhances the expansion of leukemic cells in vitro and in mice, and that this activity is dependent on its VEGFR-associated FV/FVIII domain. Finally, we show that treatment with VEGF inhibitor axitinib reduces AML cell growth and delays median leukemia latency in vivo. Our findings reveal that the NRP1/VEGF axis mediates proliferation in inv(16) AML blasts, and suggest that targeting NRP1 function could be promising in combination AML therapy.
    DOI:  https://doi.org/10.1038/s41375-024-02471-9
  5. Nat Biotechnol. 2024 Nov 22.
      Transplantation of ex vivo engineered hematopoietic stem cells (HSCs) can lead to robust clinical responses but carries risks of adverse events from bone marrow mobilization, chemotherapy conditioning and other factors. HSCs have been modified in vivo using lipid nanoparticles (LNPs) decorated with targeting moieties, which increases manufacturing complexity. Here we screen 105 LNPs without targeting ligands for effective homing to the bone marrow in mouse. We report an LNP named LNP67 that delivers mRNA to murine HSCs in vivo, primary human HSCs ex vivo and CD34+ cells in rhesus monkeys (Macaca mulatta) in vivo at doses of 0.25 and 0.4 mg kg-1. Without mobilization and conditioning, LNP67 can mediate delivery of mRNA to HSCs and their progenitor cells (HSPCs), as well as to the liver in rhesus monkeys, without serum cytokine activation. These data support the hypothesis that in vivo delivery to HSCs and HSPCs in nonhuman primates is feasible without targeting ligands.
    DOI:  https://doi.org/10.1038/s41587-024-02470-2
  6. Haematologica. 2024 Nov 21.
      Resistance to chemotherapy remains a major hurdle to the cure of Acute Myeloid Leukemia (AML) patients. Recent studies indicate a minority of malignant cells, termed drug-tolerant persisters (DTPs), stochastically upregulate stress pathways to evade cell death upon acute exposure to chemotherapy without acquiring new genetic mutations. This chemoresistant state is transient and the cells return to baseline after removal of chemotherapy. Yet, the mechanisms employed by DTPs to resist chemotherapy are not well understood and it is largely unknown whether these mechanisms are also seen in patients receiving chemotherapy. Here, we used leukemia cell lines, primary AML patient samples and samples from patients with AML receiving systemic chemotherapy to study the DTP state. We demonstrated that a subset of AML cells transiently increases membrane rigidity to resist killing due to acute exposure to Daunorubicin and Ara-C. Upon removal of the chemotherapy, membrane rigidity returned to baseline and the cells regained chemosensitivity. Although resistant to chemotherapy, the increased membrane rigidity, rendered AML cells more susceptible to T-cell mediated killing. Thus, we identified a novel mechanism by which DTP leukemic cells evade chemotherapy and a strategy to eradicate these persistent cells.
    DOI:  https://doi.org/10.3324/haematol.2024.286018
  7. Expert Rev Proteomics. 2024 Nov 22. 1-14
       INTRODUCTION: Acute myeloid leukemia (AML) is an aggressive and poor-prognosis blood cancer. Despite a low mutation burden compared to other cancers, AML is heterogenous and identifying robust therapeutic targets has been difficult. Genomic profiling has greatly advanced our understanding of AML, and has revealed targets for AML therapy. However, only 50% of AML patients have gene mutations that are currently druggable, and relapse rates remain high. The addition of proteomic profiling is emerging to address these challenges.
    AREAS COVERED: Using references collected through Pubmed, we review recent studies that have combined genomic and proteomic profiling (i.e. proteogenomic profiling), as well as studies that have additionally integrated other omics approaches, such as phosphoproteomics. We highlight how proteogenomic profiling promises to deconvolve the cellular pathways driving leukemogenesis, uncover novel therapeutic targets, and identify biomarkers of response to novel and existing therapies.
    EXPERT OPINION: Proteogenomic profiling is providing unparalleled insight into AML, and is beginning to identify robust biomarkers. Standardization of workflows will be required before mass spectrometry-based proteomic assays can be integrated into routine clinical use. However, the demonstrated ability to adapt signatures into biomarker panels that can be assayed by existing clinical workflows is enabling current clinical translation.
    Keywords:  Acute myeloid leukemia; biomarkers; phosphoproteomics; precision therapy; proteogenomics; proteomics
    DOI:  https://doi.org/10.1080/14789450.2024.2431272
  8. bioRxiv. 2024 Oct 29. pii: 2024.10.26.620318. [Epub ahead of print]
      Genetic mutations are being thoroughly mapped in human cancers, yet a fundamental question in cancer biology is whether such mutations are functionally required for cancer initiation, maintenance of established cancer, or both. Here, we study this question in the context of human acute myeloid leukemia (AML), where DNMT3AR882 missense mutations often arise early, in pre-leukemic clonal hematopoiesis, and corrupt the DNA methylation landscape to initiate leukemia. We developed CRISPR-based methods to directly correct DNMT3AR882 mutations in leukemic cells obtained from patients. Surprisingly, DNMT3AR882 mutations were largely dispensable for disease maintenance. Replacing DNMT3AR882 mutants with wild-type DNMT3A did not impair the ability of AML cells to engraft in vivo, and minimally altered DNA methylation. Taken together, DNMT3AR882 mutations are initially necessary for AML initiation, but are largely dispensable for disease maintenance. The notion that initiating oncogenes differ from those that maintain cancer has important implications for cancer evolution and therapy.
    DOI:  https://doi.org/10.1101/2024.10.26.620318
  9. Haematologica. 2024 Nov 21.
      Acquired resistance to immunomodulatory drugs (IMiDs) remains a significant unmet need in the treatment landscape of multiple myeloma (MM). CRBN pathway-dependent mechanisms are known to be vital contributors to IMiD resistance; however, they may account for only a small proportion. Recent research has unveiled additional mechanisms of acquired IMiD resistance that are independent of the CRBN pathway. In this review, we provide a comprehensive overview of the existing work on IMiD resistance in MM, focusing specifically on the emerging evidence of CRBN pathway-independent mechanisms. Finally, we discuss the plausible actionable strategies and outlook for IMiD-based therapies moving forward.
    DOI:  https://doi.org/10.3324/haematol.2024.285636
  10. Nat Commun. 2024 Nov 18. 15(1): 9964
      Cell migration through small constrictions during cancer metastasis requires significant deformation of the nucleus, with associated mechanical stress on the nuclear lamina and chromatin. However, how mechanical deformation impacts various subnuclear structures, including protein and nucleic acid-rich biomolecular condensates, is largely unknown. Here, we find that cell migration through confined spaces gives rise to mechanical deformations of the chromatin network, which cause embedded nuclear condensates, including nucleoli and nuclear speckles, to deform and coalesce. Chromatin deformations exhibit differential behavior in the advancing vs. trailing region of the nucleus, with the trailing half being more permissive for de novo condensate formation. We show that this results from increased chromatin heterogeneity, which gives rise to a shift in the binodal phase boundary. Taken together, our findings show how chromatin deformation impacts condensate assembly and properties, which can potentially contribute to cellular mechanosensing.
    DOI:  https://doi.org/10.1038/s41467-024-54120-5
  11. Methods Mol Biol. 2025 ;2871 131-143
      FOXO transcription factors respond to a number of different stresses by shuttling from the cytoplasm to the nucleus where they upregulate hundreds of target genes with diverse cellular functions. The cellular consequences of FOXO activation are both stress and cell-type specific. Recent evidence suggests that one way in which FOXO dictates stress-specific outcomes is through distinct nuclear/cytoplasmic shuttling dynamics. Here we outline methods for measuring FOXO nuclear shuttling dynamics using fluorescence-based reporters.
    Keywords:  Cell fate; FOXO; Immunofluorescence; Nuclear to cytoplasmic shuttling; Protein dynamics; Time-lapse microscopy
    DOI:  https://doi.org/10.1007/978-1-0716-4217-7_12
  12. Leukemia. 2024 Nov 17.
      Immunotherapy has demonstrated promise as a treatment for acute myeloid leukemia (AML). However, there is still an urgent need to identify new molecules that inhibit the immune response to AML. Most prior research in this area has focused on protein-protein interaction interfaces. While carbohydrates also regulate immune recognition, the role of cell-surface glycans in driving AML immune evasion is comparatively understudied. The Siglecs, for example, are an important family of inhibitory, glycan-binding signaling receptors that have emerged as prime targets for cancer immunotherapy in recent years. In this study, we find that AML cells express ligands for the receptor Siglec-9 at high levels. Integrated CRISPR genomic screening and clinical bioinformatic analysis identified ST3GAL4 as a potential driver of Siglec-9 ligand expression in AML. Depletion of ST3GAL4 by CRISPR-Cas9 knockout (KO) dramatically reduced the expression of Siglec-9 ligands in AML cells. Mass spectrometry analysis of cell-surface glycosylation in ST3GAL4 KO cells revealed that Siglec-9 primarily binds N-linked sialoglycans on these cell types. Finally, we found that ST3GAL4 KO enhanced the sensitivity of AML cells to phagocytosis by Siglec-9-expressing macrophages. This work reveals a novel axis of immune evasion and implicates ST3GAL4 as a possible target for  immunotherapy in AML.
    DOI:  https://doi.org/10.1038/s41375-024-02454-w
  13. Expert Rev Hematol. 2024 Nov 18. 1-12
       INTRODUCTION: The introduction of venetoclax has revolutionized the treatment landscape of acute myeloid leukemia, offering new therapeutic opportunities. However, the clinical response to venetoclax varies significantly between patients, with many experiencing limited duration of response.
    AREAS COVERED: Identified resistance mechanisms include both intrinsic and acquired resistance to VEN. The former is associated with cell lineage and differentiation state. The latter includes dependency on alternative BCL-2 family anti-apoptotic protein(s) mediated by genetic, epigenetic, or post-translational mechanisms, mitochondrial and metabolic involvement, as well as microenvironment. Understanding these mechanisms is crucial for optimizing venetoclax-based therapies and enhancing treatment outcomes for patients with acute myeloid leukemia. This review aims to elucidate the primary mechanisms underlying resistance to venetoclax and explore current therapeutic strategies to overcome this challenge.
    EXPERT OPINION: In patients with venetoclax resistance, alternative options include targeted combination therapies tailored to individual cases based on cytogenetics and prior treatments. Many of these therapies require further clinical investigation to validate their safety and efficacy.
    Keywords:  AML; Venetoclax; metabolism; mitochondria; resistance; signaling pathway
    DOI:  https://doi.org/10.1080/17474086.2024.2429604
  14. Mol Cell Proteomics. 2024 Nov 14. pii: S1535-9476(24)00171-3. [Epub ahead of print] 100881
      Studying regulation of protein function at a systems level necessitates an understanding of the interplay among diverse post-translational modifications (PTMs). A variety of proteomics sample processing workflows are currently used to study specific PTMs but rarely characterize multiple types of PTMs from the same sample inputs. Method incompatibilities and laborious sample preparation steps complicate large-scale physiological investigations and can lead to variations in results. The single-pot, solid-phase-enhanced sample preparation (SP3) method for sample cleanup is compatible with different lysis buffers and amenable to automation, making it attractive for high-throughput multi-PTM profiling. Herein, we describe an integrative SP3 workflow for multiplexed quantification of protein abundance, cysteine thiol oxidation, phosphorylation, and acetylation. The broad applicability of this approach is demonstrated using cell and tissue samples, and its utility for studying interacting regulatory networks is highlighted in a time-course experiment of cytokine-treated β-cells. We observed a swift response in global regulation of protein abundances consistent with rapid activation of JAK-STAT and NF-κB signaling pathways. Regulators of these pathways as well as proteins involved in their target processes displayed multi-PTM dynamics indicative of a complex cellular response stages: acute, adaptation, and chronic (prolonged stress). PARP14, a negative regulator of JAK-STAT, had multiple co-localized PTMs that may be involved in intraprotein regulatory crosstalk. Our workflow provides a high-throughput platform that can profile multi-PTMomes from the same sample set, which is valuable in unraveling the functional roles of PTMs and their co-regulation.
    Keywords:  Multi-PTM; SP3; acetylation; cysteine thiol oxidation; phosphorylation
    DOI:  https://doi.org/10.1016/j.mcpro.2024.100881
  15. Sci Rep. 2024 Nov 20. 14(1): 28805
      Multiple myeloma (MM) is a hematological malignancy originating from plasma cells. Genetically, MM is categorized into two subtypes: hyperdiploid and non-hyperdiploid tumors, with distinct chromosomal characteristics. Human myeloma cell lines (HMCLs) are instrumental in understanding MM and identifying therapeutic targets. However, their utility depends on their resemblance to patient-derived cells. We analyzed protein expression of cell surface markers, including myeloma drug targets and immunotherapy-relevant markers, in nine HMCLs using flow cytometry. RNAseq analysis was performed to correlate protein expression with transcriptomic data. While five cell surface markers (CD47, CD49d, CD138, CD269 (BCMA), and GPRC5D) were highly expressed and three (CD19, CD20, and CD117) were low or negative across cell lines, heterogenous expression was observed for the 16 remaining markers. A comparative study with patient-derived transcriptomic data indicated that four of our six in-house HMCLs closely resembled patient disease. Furthermore, our hyperdiploid HMCLs correlated better with hyperdiploid than non-hyperdiploid patient samples. Here, we also describe three previously uncharacterized HMCLs; IH-1, URVIN and FOLE. Our findings underscore the importance of evaluating surface protein expression in HMCLs when modeling MM. The observed variations in expression levels emphasize the need for a strategic selection of cell lines based on the study's objectives.
    DOI:  https://doi.org/10.1038/s41598-024-80263-y
  16. Regen Ther. 2024 Jun;26 1030-1036
      People still hold the concept of using cell-based treatments to regenerate missing neurons in high esteem. CD117+ cells are considered favorable stem cells for regenerative medicine. The objective of this research was to examine the impact of Alginate-Gelatin (Alg-Gel) hydrogel on the process of neurogenic differentiation of CD117+ cells utilizing a cytokines secretion test conducted in a laboratory setting. To achieve this objective, bone marrow-CD117+ cells were isolated using the MACS technique and then transformed into neuron cells using a neurogenic differentiation medium. The characterization of enriched CD117+ cells has been done with flow cytometry as well as immunocytochemistry. Next, the cells underwent western blotting assay to evaluate the signaling pathways. Subsequently, the culture media was obtained from both groups in order to determine cytokine levels. The study revealed that the Alg-Gel hydrogel had a notable impact on enhancing the protein expression of neuron markers such as β-tubulin and Wnt/catenin signaling pathway components in CD117+ neurogenic differentiated cells. Furthermore, the cultured medium from the experimental group exhibited a notable abundance of IL-6 and IL-10 in comparison to the control group. The observed in vitro effects of Alg-Gel hydrogel on neurogenic differentiation of CD117+ cells are likely to be caused by the cytokines that are released.
    Keywords:  Alginate-gelatin hydrogel; CD117+ hematopoietic stem cells; Neurogenic differentiation; Regenerative medicine
    DOI:  https://doi.org/10.1016/j.reth.2024.10.009