bims-scepro 2024-08-04 papers

bims-scepro

Biomed News

on Stem cell proteostasis

Issue of 2024–08–04
twenty-six papers selected by
William Grey, University of York

Bone marrow niches for hematopoietic stem cells.
CHIP: a clonal odyssey of the bone marrow niche.
The hematopoietic stem cell expansion niche in fetal liver: current state of the art and the way forward.
Characterization of Multicellular Niches Supporting Hematopoietic Stem Cells Within Distinct Zones.
Adhesion GPCR ADGRE2 Maintains Proteostasis to Promote Progression in Acute Myeloid Leukemia.
HES1 is required for mouse fetal hematopoiesis.
Baf155 controls hematopoietic differentiation and regeneration through chromatin priming.
A Single Trophoblast Layer Acts as the Gatekeeper at the Endothelial-Hematopoietic Crossroad in the Placenta.
Microenvironment matters: In vitro 3D bone marrow niches differentially modulate survival, phenotype and drug responses of acute myeloid leukemia (AML) cells.
STAG2 mutations reshape the cohesin-structured spatial chromatin architecture to drive gene regulation in acute myeloid leukemia.
Exploring non-viral methods for the delivery of CRISPR-Cas ribonucleoprotein to hematopoietic stem cells.
Limiting the impact of protein leakage in single-cell proteomics.
FLT3-PROTACs for combating AML resistance: Analytical overview on chimeric agents developed, challenges, and future perspectives.
Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells.
Investigational CXCR4 inhibitors in early phase development for the treatment of hematological malignancies.
mTORC1 activity oscillates throughout the cell cycle, promoting mitotic entry and differentially influencing autophagy induction.
Discovery of novel penetrating peptides able to target human leukemia and lymphoma for enhanced PROTAC delivery.
Clonal analysis of fetal hematopoietic stem/progenitor cells reveals how post-transplantation capabilities are distributed.
The E3 ubiquitin ligase Herc1 modulates the response to nucleoside analogs in acute myeloid leukemia.
PDLIM2 is a novel E5 ubiquitin ligase enhancer that stabilizes ROC1 and recruits the ROC1-SCF ubiquitin ligase to ubiquitinate and degrade NF-κB RelA.
Machine learning-based biomarker screening for acute myeloid leukemia prognosis and therapy from diverse cell-death patterns.
Protocol for differentiation of monocytes and macrophages from human induced pluripotent stem cells.
Biomineralization-Tuned Nanounits Reprogram the Signal Transducer and Activator of Transcription 3 Signaling for Ferroptosis-Immunotherapy in Cancer Stem Cells.
Caspase-2 is essential for proliferation and self-renewal of nucleophosmin-mutated acute myeloid leukemia.
Immune infiltration-related genes regulate the progression of AML by invading the bone marrow microenvironment.
Gilteritinib Reduces FLT3 Expression in Acute Myeloid Leukemia Cells.

Hemasphere. 2024 Aug;8(8): e133

Bone marrow niches for hematopoietic stem cells.

Ana Luísa Pereira, Serena Galli, César Nombela-Arrieta.

Hematopoietic stem cells (HSCs) are the cornerstone of the hematopoietic system. HSCs sustain the continuous generation of mature blood derivatives while self-renewing to preserve a relatively constant pool of progenitors throughout life. Yet, long-term maintenance of functional HSCs exclusively takes place in association with their native tissue microenvironment of the bone marrow (BM). HSCs have been long proposed to reside in fixed and identifiable anatomical units found in the complex BM tissue landscape, which control their identity and fate in a deterministic manner. In the last decades, tremendous progress has been made in the dissection of the cellular and molecular fabric of the BM, the structural organization governing tissue function, and the plethora of interactions established by HSCs. Nonetheless, a holistic model of the mechanisms controlling HSC regulation in their niche is lacking to date. Here, we provide an overview of our current understanding of BM anatomy, HSC localization, and crosstalk within local cellular neighborhoods in murine and human tissues, and highlight fundamental open questions on how HSCs functionally integrate in the BM microenvironment.

DOI: https://doi.org/10.1002/hem3.133
J Clin Invest. 2024 Aug 01. pii: e180068. [Epub ahead of print]134(15):

CHIP: a clonal odyssey of the bone marrow niche.

Wolfgang E Schleicher, Bridget Hoag, Marco De Dominici, James DeGregori, Eric M Pietras.

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by the selective expansion of hematopoietic stem and progenitor cells (HSPCs) carrying somatic mutations. While CHIP is typically asymptomatic, it has garnered substantial attention due to its association with the pathogenesis of multiple disease conditions, including cardiovascular disease (CVD) and hematological malignancies. In this Review, we will discuss seminal and recent studies that have advanced our understanding of mechanisms that drive selection for mutant HSPCs in the BM niche. Next, we will address recent studies evaluating potential relationships between the clonal dynamics of CHIP and hematopoietic development across the lifespan. Next, we will examine the roles of systemic factors that can influence hematopoietic stem cell (HSC) fitness, including inflammation, and exposures to cytotoxic agents in driving selection for CHIP clones. Furthermore, we will consider how - through their impact on the BM niche - lifestyle factors, including diet, exercise, and psychosocial stressors, might contribute to the process of somatic evolution in the BM that culminates in CHIP. Finally, we will review the role of old age as a major driver of selection in CHIP.

DOI: https://doi.org/10.1172/JCI180068
Exp Hematol. 2024 Jul 26. pii: S0301-472X(24)00444-2. [Epub ahead of print] 104585

The hematopoietic stem cell expansion niche in fetal liver: current state of the art and the way forward.

Harsh Agrawal, Shubham Haribhau Mehatre, Satish Khurana.

Hematopoietic development goes through a number of embryonic sites that host hematopoietic progenitor and stem cells with function required at specific developmental stages. Among embryonic sites, fetal liver (FL) hosts definitive hematopoietic stem cells (HSCs) capable of engrafting adult hematopoietic system and supports their rapid expansion. Hence, this site provides an excellent model to understand the cellular and molecular components of the machinery involved in HSC proliferative events leading to their overall expansion. It has been unequivocally established that extrinsic regulators orchestrate events that maintain HSC function. While most studies on extrinsic regulation of HSC function are targeted at adult bone marrow (BM) hematopoiesis, little is known about how FL HSC function is regulated by their micro-niche. This review provides the current state of our understanding on molecular and cellular niche factors that support FL hematopoiesis.

DOI: https://doi.org/10.1016/j.exphem.2024.104585
bioRxiv. 2024 Jul 17. pii: 2024.06.28.601225. [Epub ahead of print]

Characterization of Multicellular Niches Supporting Hematopoietic Stem Cells Within Distinct Zones.

Ruochen Dong, Hua Li, Xi C He, Chen Wang, Anoja Perera, Seth Malloy, Jonathon Russell, Wenting Li, Kaitlyn Petentler, Xinjian Mao, Zhe Yang, Michael Epp, Kate Hall, Allison Scott, Mary C McKinney, Shengping Huang, Sarah E Smith, Mark Hembree, Yongfu Wang, Zulin Yu, Jeffery S Haug, Jay Unruh, Brian Slaughter, Xunlei Kang, Linheng Li.

Previous studies of hematopoietic stem cells (HSCs) primarily focused on single cell-based niche models, yielding fruitful but conflicting findings 1-5 . Here we report our investigation on the fetal liver (FL) as the primary fetal hematopoietic site using spatial transcriptomics. Our study reveals two distinct niches: the portal-vessel (PV) niche and the sinusoidal niche. The PV niche, composing N-cadherin (N-cad) Hi Pdgfrα + mesenchymal stromal cells (MSCs), endothelial cells (ECs), and N-cad Lo Albumin + hepatoblasts, maintains quiescent and multipotential FL-HSCs. Conversely, the sinusoidal niche, comprising ECs, hepatoblasts and hepatocytes, as well as potential macrophages and megakaryocytes, supports proliferative FL-HSCs biased towards myeloid lineages. Unlike prior reports on the role of Cxcl12, with its depletion from vessel-associated stromal cells leading to 80% of HSCs' reduction in the adult bone marrow (BM) 6,7 , depletion of Cxcl12 via Cdh2 CreERT (encoding N-cad) induces altered localization of HSCs from the PV to the sinusoidal niches, resulting in an increase of HSC number but with myeloid-bias. Similarly, we discovered that adult BM encompasses two niches within different zones, each composed of multi-cellular components: trabecular bone area (TBA, or metaphysis) supporting deep-quiescent HSCs, and central marrow (CM, or diaphysis) fostering heterogenous proliferative HSCs. This study transforms our understanding of niches by shifting from single cell-based to multicellular components within distinct zones, illuminating the intricate regulation of HSCs tailored to their different cycling states.

DOI: https://doi.org/10.1101/2024.06.28.601225
Cancer Res. 2024 Jul 02. 84(13): 2090-2108

Adhesion GPCR ADGRE2 Maintains Proteostasis to Promote Progression in Acute Myeloid Leukemia.

Deyu Huang, Zebin Yu, Huan Lu, Penglei Jiang, Xinyue Qian, Yingli Han, Pengxu Qian.

Acute myeloid leukemia (AML) is an aggressive and heterogeneous hematologic malignancy. In elderly patients, AML incidence is high and has a poor prognosis due to a lack of effective therapies. G protein-coupled receptors (GPCR) play integral roles in physiologic processes and human diseases. Particularly, one third of adhesion GPCRs, the second largest group of GPCRs, are highly expressed in hematopoietic stem and progenitor cells or lineage cells. Here, we investigate the role of adhesion GPCRs in AML and whether they could be harnessed as antileukemia targets. Systematic screening of the impact of adhesion GPCRs on AML functionality by bioinformatic and functional analyses revealed high expression of ADGRE2 in AML, particularly in leukemic stem cells, which is associated with poor patient outcomes. Silencing ADGRE2 not only exerts antileukemic effects in AML cell lines and cells derived from patients with AML in vitro, but also delays AML progression in xenograft models in vivo. Mechanistically, ADGRE2 activates phospholipase Cβ/protein kinase C/MEK/ERK signaling to enhance the expression of AP1 and transcriptionally drive the expression of DUSP1, a protein phosphatase. DUSP1 dephosphorylates Ser16 in the J-domain of the co-chaperone DNAJB1, which facilitates the DNAJB1-HSP70 interaction and maintenance of proteostasis in AML. Finally, combined inhibition of MEK, AP1, and DUSP1 exhibits robust therapeutic efficacy in AML xenograft mouse models. Collectively, this study deciphers the roles and mechanisms of ADGRE2 in AML and provides a promising therapeutic strategy for treating AML. Significance: Increased expression of the adhesion GPCR member ADGRE2 in AML supports leukemia stem cell self-renewal and leukemogenesis by modulating proteostasis via an MEK/AP1/DUSP1 axis, which can be targeted to suppress AML progression.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-2314
Stem Cell Res Ther. 2024 Jul 29. 15(1): 235

HES1 is required for mouse fetal hematopoiesis.

Anthony Z Zhu, Zhilin Ma, Emily V Wolff, Zichen Lin, Zhenxia J Gao, Xue Li, Wei Du.

   BACKGROUND: Hematopoiesis in mammal is a complex and highly regulated process in which hematopoietic stem cells (HSCs) give rise to all types of differentiated blood cells. Previous studies have shown that hairy and enhancer of split (HES) repressors are essential regulators of adult HSC development downstream of Notch signaling.
METHODS: In this study, we investigated the role of HES1, a member of HES family, in fetal hematopoiesis using an embryonic hematopoietic specific Hes1 conditional knockout mouse model by using phenotypic flow cytometry, histopathology analysis, and functional in vitro colony forming unit (CFU) assay and in vivo bone marrow transplant (BMT) assay.
RESULTS: We found that loss of Hes1 in early embryonic stage leads to smaller embryos and fetal livers, decreases hematopoietic stem progenitor cell (HSPC) pool, results in defective multi-lineage differentiation. Functionally, fetal hematopoietic cells deficient for Hes1 exhibit reduced in vitro progenitor activity and compromised in vivo repopulation capacity in the transplanted recipients. Further analysis shows that fetal hematopoiesis defects in Hes1fl/flFlt3Cre embryos are resulted from decreased proliferation and elevated apoptosis, associated with de-repressed HES1 targets, p27 and PTEN in Hes1-KO fetal HSPCs. Finally, pharmacological inhibition of p27 or PTEN improves fetal HSPCs function both in vitro and in vivo.
CONCLUSION: Together, our findings reveal a previously unappreciated role for HES1 in regulating fetal hematopoiesis, and provide new insight into the differences between fetal and adult HSC maintenance.

Keywords:  Apoptosis; Fetal hematopoiesis; Fetal liver; HES1; Hematopoietic stem progenitor cells; Stem cell quiescence

DOI:  https://doi.org/10.1186/s13287-024-03836-8
Cell Rep. 2024 Jul 30. pii: S2211-1247(24)00887-8. [Epub ahead of print]43(8): 114558

Baf155 controls hematopoietic differentiation and regeneration through chromatin priming.

Jun Wu, Changxu Fan, Ashraf Ul Kabir, Karen Krchma, Minseo Kim, Yoojung Kwon, Xiaoyun Xing, Ting Wang, Kyunghee Choi.

  Chromatin priming promotes cell-type-specific gene expression, lineage differentiation, and development. The mechanism of chromatin priming has not been fully understood. Here, we report that mouse hematopoietic stem and progenitor cells (HSPCs) lacking the Baf155 subunit of the BAF (BRG1/BRM-associated factor) chromatin remodeling complex produce a significantly reduced number of mature blood cells, leading to a failure of hematopoietic regeneration upon transplantation and 5-fluorouracil (5-FU) injury. Baf155-deficient HSPCs generate particularly fewer neutrophils, B cells, and CD8+ T cells at homeostasis, supporting a more immune-suppressive tumor microenvironment and enhanced tumor growth. Single-nucleus multiomics analysis reveals that Baf155-deficient HSPCs fail to establish accessible chromatin in selected regions that are enriched for putative enhancers and binding motifs of hematopoietic lineage transcription factors. Our study provides a fundamental mechanistic understanding of the role of Baf155 in hematopoietic lineage chromatin priming and the functional consequences of Baf155 deficiency in regeneration and tumor immunity.

Keywords:  Baf155; CP: Molecular biology; CP: Stem cell research; Smarcc1; Srg3; chromatin priming; hematopoietic lineage differentiation; hematopoietic regeneration; hematopoietic stem cell; tumor immunity

DOI:  https://doi.org/10.1016/j.celrep.2024.114558
bioRxiv. 2024 Jul 16. pii: 2024.07.12.603303. [Epub ahead of print]

A Single Trophoblast Layer Acts as the Gatekeeper at the Endothelial-Hematopoietic Crossroad in the Placenta.

Pratik Home, Ananya Ghosh, Ram Parikshan Kumar, Soma Ray, Sumedha Gunewardena, Rajnish Kumar, Purbasa Dasgupta, Namrata Roy, Abhik Saha, Madhu M Ouseph, Gustavo W Leone, Soumen Paul.

During embryonic development the placental vasculature acts as a major hematopoietic niche, where endothelial to hematopoietic transition ensures emergence of hematopoietic stem cells (HSCs). However, the molecular mechanisms that regulate the placental hematoendothelial niche are poorly understood. Using a parietal trophoblast giant cell (TGC)-specific knockout mouse model and single-cell RNA-sequencing, we show that the paracrine factors secreted by the TGCs are critical in the development of this niche. Disruptions in the TGC-specific paracrine signaling leads to the loss of HSC population and the concomitant expansion of a KDR+/DLL4+/PROM1+ hematoendothelial cell-population in the placenta. Combining single-cell transcriptomics and receptor-ligand pair analyses, we also define the parietal TGC-dependent paracrine signaling network and identify Integrin signaling as a fundamental regulator of this process. Our study elucidates novel mechanisms by which non-autonomous signaling from the primary parietal TGCs maintain the delicate placental hematopoietic-angiogenic balance and ensures embryonic and extraembryonic development.

DOI: https://doi.org/10.1101/2024.07.12.603303
Biomaterials. 2024 Jul 27. pii: S0142-9612(24)00253-9. [Epub ahead of print]312 122719

Microenvironment matters: In vitro 3D bone marrow niches differentially modulate survival, phenotype and drug responses of acute myeloid leukemia (AML) cells.

Hoi Lam Cheung, Yu Hin Wong, Yuk Yin Li, Xingxing Yang, Lok Him Ko, Jessica Evangeline Tan Kabigting, Koon Chuen Chan, Anskar Yu Hung Leung, Barbara Pui Chan.

  Acute myeloid leukemia (AML) is a deadly form of leukemia with ineffective traditional treatment and frequent chemoresistance-associated relapse. Personalized drug screening holds promise in identifying optimal regimen, nevertheless, primary AML cells undergo spontaneous apoptosis during cultures, invalidating the drug screening results. Here, we reconstitute a 3D osteogenic niche (3DON) mimicking that in bone marrow to support primary AML cell survival and phenotype maintenance in cultures. Specifically, 3DON derived from osteogenically differentiated mesenchymal stem cells (MSC) from healthy and AML donors are co-cultured with primary AML cells. The AML cells under the AML_3DON niche showed enhanced viability, reduced apoptosis and maintained CD33+ CD34-phenotype, associating with elevated secretion of anti-apoptotic cytokines in the AML_3DON niche. Moreover, AML cells under the AML_3DON niche exhibited low sensitivity to two FDA-approved chemotherapeutic drugs, further suggesting the physiological resemblance of the AML_3DON niche. Most interestingly, AML cells co-cultured with the healthy_3DON niche are highly sensitive to the same sample drugs. This study demonstrates the differential responses of AML cells towards leukemic and healthy bone marrow niches, suggesting the impact of native cancer cell niche in drug screening, and the potential of re-engineering healthy bone marrow niche in AML patients as chemotherapeutic adjuvants overcoming chemoresistance, respectively.

Keywords:  3D osteogenic niche; AML; Cancer microenvironment; Personalized drug screening

DOI:  https://doi.org/10.1016/j.biomaterials.2024.122719
Cell Rep. 2024 Jul 25. pii: S2211-1247(24)00827-1. [Epub ahead of print] 114498

STAG2 mutations reshape the cohesin-structured spatial chromatin architecture to drive gene regulation in acute myeloid leukemia.

Alexander Fischer, Benjamín Hernández-Rodríguez, Roger Mulet-Lazaro, Margit Nuetzel, Fabian Hölzl, Stanley van Herk, François G Kavelaars, Hanna Stanewsky, Ute Ackermann, Amadou H Niang, Noelia Diaz, Edith Reuschel, Nicholas Strieder, Inmaculada Hernández-López, Peter J M Valk, Juan M Vaquerizas, Michael Rehli, Ruud Delwel, Claudia Gebhard.

  Cohesin shapes the chromatin architecture, including enhancer-promoter interactions. Its components, especially STAG2, but not its paralog STAG1, are frequently mutated in myeloid malignancies. To elucidate the underlying mechanisms of leukemogenesis, we comprehensively characterized genetic, transcriptional, and chromatin conformational changes in acute myeloid leukemia (AML) patient samples. Specific loci displayed altered cohesin occupancy, gene expression, and local chromatin activation, which were not compensated by the remaining STAG1-cohesin. These changes could be linked to disrupted spatial chromatin looping in cohesin-mutated AMLs. Complementary depletion of STAG2 or STAG1 in primary human hematopoietic progenitors (HSPCs) revealed effects resembling STAG2-mutant AML-specific changes following STAG2 knockdown, not invoked by the depletion of STAG1. STAG2-deficient HSPCs displayed impaired differentiation capacity and maintained HSPC-like gene expression. This work establishes STAG2 as a key regulator of chromatin contacts, gene expression, and differentiation in the hematopoietic system and identifies candidate target genes that may be implicated in human leukemogenesis.

Keywords:  CP: Cancer; CP: Molecular biology; Hi-C; RAD21; STAG2; acute myeloid leukemia; cohesin mutations; epigenetics; gene regulation; hematopoiesis; hematopoietic stem and progenitor cells; spatial chromatin structure

DOI:  https://doi.org/10.1016/j.celrep.2024.114498
Stem Cell Res Ther. 2024 Jul 29. 15(1): 233

Exploring non-viral methods for the delivery of CRISPR-Cas ribonucleoprotein to hematopoietic stem cells.

Zahra Molaei, Zahra Jabbarpour, Azadeh Omidkhoda, Naser Ahmadbeigi.

  Gene manipulation of hematopoietic stem cells (HSCs) using the CRISPR/Cas system as a potent genome editing tool holds immense promise for addressing hematologic disorders. An essential hurdle in advancing this treatment lies in effectively delivering CRISPR/Cas to HSCs. While various delivery formats exist, Ribonucleoprotein complex (RNP) emerges as a particularly efficient option. RNP complexes offer enhanced gene editing capabilities, devoid of viral vectors, with rapid activity and minimized off-target effects. Nevertheless, novel delivery methods such as microfluidic-based techniques, filtroporation, nanoparticles, and cell-penetrating peptides are continually evolving. This study aims to provide a comprehensive review of these methods and the recent research on delivery approaches of RNP complexes to HSCs.

Keywords:  CRISPR-Cas System; Delivery strategies; Genetic therapy; Genome editing; Hematopoietic stem cell

DOI:  https://doi.org/10.1186/s13287-024-03848-4
bioRxiv. 2024 Jul 27. pii: 2024.07.26.605378. [Epub ahead of print]

Limiting the impact of protein leakage in single-cell proteomics.

Andrew Leduc, Yanxin Xu, Gergana Shipkovenska, Zhixun Dou, Nikolai Slavov.

Limiting artifacts during sample preparation can significantly increase data quality in single-cell proteomics experiments. Towards this goal, we characterize the impact of protein leakage by analyzing thousands of primary single cells that were prepared either fresh immediately after dissociation or cryopreserved and prepared at a later date. We directly identify permeabilized cells and use the data to define a signature for protein leakage. We use this signature to build a classifier for identifying damaged cells that performs accurately across cell types and species.

DOI: https://doi.org/10.1101/2024.07.26.605378
Eur J Med Chem. 2024 Aug 01. pii: S0223-5234(24)00598-1. [Epub ahead of print]277 116717

FLT3-PROTACs for combating AML resistance: Analytical overview on chimeric agents developed, challenges, and future perspectives.

Heba M Hesham, Eman M E Dokla, Eman Z Elrazaz, Deena S Lasheen, Dalal A Abou El Ella.

  The urgent and unmet medical demand of acute myeloid leukemia (AML) patients has driven the drug discovery process for expansion of the landscape of AML treatment. Despite the several agents developed for treatment of AML, more than 60 % of treated patients undergo relapse again after re-emission, thus, no complete cure for this complex disease has been reached yet. Targeted oncoprotein degradation is a new paradigm that can be employed to solve drug resistance, disease relapse, and treatment failure in complex diseases as AML, the most lethal hematological malignancy. AML is an aggressive blood cancer form and the most common type of acute leukemia, with bad outcomes and a very poor 5-year survival rate. FLT3 mutations occur in about 30 % of AML cases and FLT3-ITD is associated with poor prognosis of this disease. Prevalent FLT3 mutations include internal tandem duplication and point mutations (e.g., D835) in the tyrosine kinase domain, which induce FLT3 kinase activation and result in survival and proliferation of AML cells again. Currently approved FLT3 inhibitors suffer from limited clinical efficacy due to FLT3 reactivation by mutations, therefore, alternative new treatments are highly needed. Proteolysis-targeting chimera (PROTAC) is a bi-functional molecule that consists of a ligand of the protein of interest, FLT3 inhibitor in our case, that is covalently linked to an E3 ubiquitin ligase ligand. Upon FLT3-specific PROTAC binding to FLT3, the PROTAC can recruit E3 for FLT3 ubiquitination, which is subsequently subjected to proteasome-mediated degradation. In this review we tried to address the question if PROTAC technology has succeeded in tackling the disease relapse and treatment failure of AML. Next, we explored the latest FLT3-targeting PROTACs developed in the past few years such as quizartinib-based PROTACs, dovitinib-based PROTACs, gilteritinib-based PROTACs, and others. Then, we followed with a deep analysis of their advantages regarding potency improvement and overcoming AML drug resistance. Finally, we discussed the challenges facing these chimeric molecules with proposed future solutions to circumvent them.

Keywords:  Acute myeloid leukemia; Anticancer; Drug resistance; E3 ligase; FLT3 kinase; PROTACs

DOI:  https://doi.org/10.1016/j.ejmech.2024.116717
bioRxiv. 2024 Jul 16. pii: 2024.07.14.603391. [Epub ahead of print]

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells.

Srishti U Sahu, Madalena Castro, Joseph J Muldoon, Kunica Asija, Stacia K Wyman, Netravathi Krishnappa, Justin Eyquem, David N Nguyen, Ross C Wilson.

Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in, and base editing. PERC involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating the formulation with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) bearing homology-directed repair template DNA may be included. In contrast to electroporation, PERC is appealing as it requires no dedicated hardware and has less impact on cell phenotype and viability. Due to the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Here we report methods for improved PERC-mediated editing of T cells as well as novel methods for PERC-mediated editing of HSPCs, including knockout and precise knock-in. Editing efficiencies can surpass 90% using either Cas9 or Cas12a in primary T cells or HSPCs. Because PERC calls for only three readily available reagents - protein, RNA, and peptide - and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes this approach appealing for rapid deployment in research and clinical settings.

DOI: https://doi.org/10.1101/2024.07.14.603391
Expert Opin Investig Drugs. 2024 Aug 03.

Investigational CXCR4 inhibitors in early phase development for the treatment of hematological malignancies.

Enrica Antonia Martino, Antonella Bruzzese, Caterina Labanca, Francesco Mendicino, Eugenio Lucia, Virginia Olivito, Gaia Stanzione, Annamaria Zimbo, Stefano Pozzi, Antonino Neri, Fortunato Morabito, Ernesto Vigna, Massimo Gentile.

   INTRODUCTION: CXCR4/CXCL12 axis regulates cell proliferation, survival, and differentiation, as well as the homing and mobilization of hematopoietic stem cells (HSCs) from bone marrow niches to the peripheral blood. Furthermore, CXCR4 and CXCL12 are key mediators of cross-talk between hematological malignancies and their microenvironments. CXCR4 overexpression drives disease progression, boosts tumor cell survival, and promotes chemoresistance, leading to poor prognosis.
AREAS COVERED: In light of these discoveries, scientific investigations and clinical trials have underscored the therapeutic promise found in small-molecule antagonists like plerixafor, peptides/peptidomimetics such as BKT140, monoclonal antibodies like PF-06747143 and ulocuplumab, as well as microRNAs. Their efficacy is evident in reducing tumor burden, inducing apoptosis, and sensitizing malignant cells to conventional chemotherapies. This overview delves into the pathogenic role of the CXC4/CXCL12 axis in hematological neoplasms and examines the clinical application of key CXCR4 antagonists.
EXPERT OPINION: The information collectively emphasizes the potential of CXCR4 antagonists as a therapeutic strategy for hematologic malignancies, showcasing advancements in preclinical and clinical studies. As these therapeutic strategies progress through clinical trials, their potential to reshape the prognosis of hematologic malignancies will become increasingly apparent.

Keywords:  CXCL12; CXCR4; hematological malignancies; therapy

DOI:  https://doi.org/10.1080/13543784.2024.2388567
Cell Rep. 2024 Jul 26. pii: S2211-1247(24)00872-6. [Epub ahead of print]43(8): 114543

mTORC1 activity oscillates throughout the cell cycle, promoting mitotic entry and differentially influencing autophagy induction.

Jay N Joshi, Ariel D Lerner, Frank Scallo, Alexandra N Grumet, Paul Matteson, James H Millonig, Alexander J Valvezan.

  Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a master metabolic regulator that is active in nearly all proliferating eukaryotic cells; however, it is unclear whether mTORC1 activity changes throughout the cell cycle. We find that mTORC1 activity oscillates from lowest in mitosis/G1 to highest in S/G2. The interphase oscillation is mediated through the TSC complex but is independent of major known regulatory inputs, including Akt and Mek/Erk signaling. By contrast, suppression of mTORC1 activity in mitosis does not require the TSC complex. mTORC1 has long been known to promote progression through G1. We find that mTORC1 also promotes progression through S and G2 and is important for satisfying the Chk1/Wee1-dependent G2/M checkpoint to allow entry into mitosis. We also find that low mTORC1 activity in G1 sensitizes cells to autophagy induction in response to partial mTORC1 inhibition or reduced nutrient levels. Together, these findings demonstrate that mTORC1 is differentially regulated throughout the cell cycle, with important phase-specific consequences for proliferating cells.

Keywords:  CDK1; CP: Cell biology; G2/M checkpoint; TSC complex; TSC2; autophagy; cell cycle; mTOR; mTORC1; mitosis

DOI:  https://doi.org/10.1016/j.celrep.2024.114543
Eur J Med Chem. 2024 Jul 31. pii: S0223-5234(24)00615-9. [Epub ahead of print]277 116734

Discovery of novel penetrating peptides able to target human leukemia and lymphoma for enhanced PROTAC delivery.

Qingqing Zhang, Yuying Liu, Jie Zhang, Yanchen Li, Jin Wang, Nanxin Liu, Jie Zhang, Xiaoyan Pan.

  Proteolysis targeting chimeras (PROTAC) are bifunctional chimeric molecules capable of directly degrading binding proteins through the ubiquitin-proteasome pathway. PROTACs have demonstrated significant potential in overcoming drug resistance and targeting previously untreatable targets. However, several limitations still need to be addressed, including their high molecular weight resulting in poor membrane permeability and bioavailability. In this study, we proposed that cancer-targeted penetrating peptides could enhance the cell permeability of PROTACs. We developed 26 novel targeted penetrating peptides for leukemia and lymphoma cells, among which C9C-f(3Bta) and Cyclo-C9C-R exhibited superior membrane permeability, targetability, and stability. By combining C9C-f(3Bta) and Cyclo-C9C-R with IMA-PROTAC, we effectively enhanced the anti-proliferative activity of IMA-PROTAC, facilitated degradation of Bcr-Abl protein in K562 cells, and reduced downstream STAT5 phosphorylation. Furthermore, the combined application promoted cell apoptosis while blocking G1 phase progression. HPLC-MRM-MS revealed that the combination of C9C-f(3Bta) or Cyclo-C9C-R with IMA-PROTAC significantly enhanced intracellular IMA-PROTAC content. In summary, our proof-of-concept study validated the hypothesis that combining PROTACs with targeted penetrating peptides can improve protein degradation efficiency as well as anti-proliferative capabilities.

Keywords:  Antitumor; Cancer-targeted penetrating peptide (CTP); Leukemia treatment; PROTAC; Targeted delivery

DOI:  https://doi.org/10.1016/j.ejmech.2024.116734
Stem Cell Reports. 2024 Jul 24. pii: S2213-6711(24)00212-1. [Epub ahead of print]

Clonal analysis of fetal hematopoietic stem/progenitor cells reveals how post-transplantation capabilities are distributed.

Olivia J Stonehouse, Christine Biben, Tom S Weber, Alexandra Garnham, Katie A Fennell, Alison Farley, Antoine F Terreaux, Warren S Alexander, Mark A Dawson, Shalin H Naik, Samir Taoudi.

It has been proposed that adult hematopoiesis is sustained by multipotent progenitors (MPPs) specified during embryogenesis. Adult-like hematopoietic stem cell (HSC) and MPP immunophenotypes are present in the fetus, but knowledge of their functional capacity is incomplete. We found that fetal MPP populations were functionally similar to adult cells, albeit with some differences in lymphoid output. Clonal assessment revealed that lineage biases arose from differences in patterns of single-/bi-lineage differentiation. Long-term (LT)- and short-term (ST)-HSC populations were distinguished from MPPs according to capacity for clonal multilineage differentiation. We discovered that a large cohort of long-term repopulating units (LT-RUs) resides within the ST-HSC population; a significant portion of these were labeled using Flt3-cre. This finding has two implications: (1) use of the CD150+ LT-HSC immunophenotype alone will significantly underestimate the size and diversity of the LT-RU pool and (2) LT-RUs in the ST-HSC population have the attributes required to persist into adulthood.

DOI: https://doi.org/10.1016/j.stemcr.2024.07.003
Blood Adv. 2024 Aug 02. pii: bloodadvances.2023011540. [Epub ahead of print]

The E3 ubiquitin ligase Herc1 modulates the response to nucleoside analogs in acute myeloid leukemia.

Maja Jankovic, William Wl Poon, Cristobal Gonzales-Losada, Gabriela Galicia Vazquez, Bahram Sharif-Askari, Yi Ding, Constance Craplet-Desombre, Alexandru Ilie, Jiantao Shi, Yongie Wang, Ashok Kumar Jayavelu, Alexandre Orthwein, Francois Emile Mercier.

For several decades, induction therapy with nucleoside analogs, in particular cytarabine (Ara-C) and, to a lesser extent, fludarabine, has been the standard of care for patients diagnosed with acute myeloid leukemia (AML). Still, the anti-tumor efficacy of nucleoside analogs is often limited by intrinsic and acquired drug resistance, thereby leading to poor therapeutic response and suboptimal clinical outcomes. Here, we used genome-wide CRISPR-based pharmacogenomic screening to map the genetic factors that modulate the response to nucleoside analogs in AML and identified the E3 ubiquitin ligase Herc1 as a key modulator of Ara-C response in the MLL/AF9 (MA) and the HOXA9/MEIS1 (HM) murine AML models both in vitro and in vivo. Loss of HERC1 enhanced nucleoside analog-induced cell death in both murine and human AML cell lines by compromising cell cycle progression. In-depth proteomic analysis and subsequent validation identified deoxycytidine kinase Dck as a novel target of Herc1 in MA and HM murine cells. We observed that HERC1 is overexpressed in AML compared to other cancer types and higher HERC1 expression is associated with shorter overall survival of patients with AML in the TCGA and BEAT-AML cohorts. Collectively, this study highlights the importance of HERC1 in the response of AML cells to nucleoside analogs, thereby establishing this E3 ubiquitin ligase as a novel predictive biomarker and potential therapeutic target for the treatment of AML.

DOI: https://doi.org/10.1182/bloodadvances.2023011540
Cell Biosci. 2024 Jul 30. 14(1): 99

PDLIM2 is a novel E5 ubiquitin ligase enhancer that stabilizes ROC1 and recruits the ROC1-SCF ubiquitin ligase to ubiquitinate and degrade NF-κB RelA.

Fan Sun, Gutian Xiao, Zhaoxia Qu.

  The PDZ-LIM domain-containing protein PDLIM2 is a common tumor suppressor and a key immune modulator. One main function of PDLIM2 is to promote the ubiquitination and proteasomal degradation of nuclear activated NF-κB RelA, a physiologically indispensable transcription factor whose persistent activation has been linked to almost all cancer types and inflammation-associated diseases. However, it remains unknown how PDLIM2 exerts this physiologically and pathogenically important function. Here, we show that PDLIM2 acts as a ubiquitin ligase enhancer, termed E5. It stabilizes ROC1, an essential component of SKP1/Cullin/F-box protein (SCF) ubiquitin ligases, and chaperones the ROC1-SCFβ-TrCP ubiquitin ligase to ubiquitinate nuclear RelA for proteasomal degradation in the nucleus. Consistently, silencing of ROC1, Cullin 1 or the F-box protein β-TrCP blocks RelA ubiquitination and degradation by PDLIM2. These data provide new mechanistic insights into how PDLIM2 promotes nuclear RelA ubiquitination and degradation, thereby serving as a critical tumor suppressor and a vital immune regulator. They also improve our understanding of the complex cascade of the ubiquitination and NF-κB pathways, particularly given the well-known role of the ROC1-SCFβ-TrCP ubiquitin ligase in initiating NF-κB activation by directly binding to and ubiquitinating NF-κB inhibitors for the proteasomal degradation in the cytoplasm.

Keywords:  E3 ubiquitin ligase; E5 ubiquitin ligase enhancer; NF-κB; PDLIM2; Proteasomal degradation; RBX1/ROC1; RBX1/ROC1 stabilization; RelA/p65; SCFβ-TRCP E3 ubiquitin ligase; Ubiquitination

DOI:  https://doi.org/10.1186/s13578-024-01281-x
Sci Rep. 2024 Aug 02. 14(1): 17874

Machine learning-based biomarker screening for acute myeloid leukemia prognosis and therapy from diverse cell-death patterns.

Yu Qin, Xuexue Pu, Dingtao Hu, Mingzhen Yang.

  Acute myeloid leukemia (AML) exhibits pronounced heterogeneity and chemotherapy resistance. Aberrant programmed cell death (PCD) implicated in AML pathogenesis suggests PCD-related signatures could serve as biomarkers to predict clinical outcomes and drug response. We utilized 13 PCD pathways, including apoptosis, pyroptosis, ferroptosis, autophagy, necroptosis, cuproptosis, parthanatos, entotic cell death, netotic cell death, lysosome-dependent cell death, alkaliptosis, oxeiptosis, and disulfidptosis to develop predictive models based on 73 machine learning combinations from 10 algorithms. Bulk RNA-sequencing, single-cell RNA-sequencing transcriptomic data, and matched clinicopathological information were obtained from the TCGA-AML, Tyner, and GSE37642-GPL96 cohorts. These datasets were leveraged to construct and validate the models. Additionally, in vitro experiments were conducted to substantiate the bioinformatics findings. The machine learning approach established a 6-gene pan-programmed cell death-related genes index (PPCDI) signature. Validation in two external cohorts showed high PPCDI associated with worse prognosis in AML patients. Incorporating PPCDI with clinical variables, we constructed several robust prognostic nomograms that accurately predicted prognosis of AML patients. Multi-omics analysis integrating bulk and single-cell transcriptomics revealed correlations between PPCDI and immunological features, delineating the immune microenvironment landscape in AML. Patients with high PPCDI exhibited resistance to conventional chemotherapy like doxorubicin but retained sensitivity to dasatinib and methotrexate (FDA-approved drugs for other leukemias), suggesting the potential of PPCDI to guide personalized therapy selection in AML. In summary, we developed a novel PPCDI model through comprehensive analysis of diverse programmed cell death pathways. This PPCDI signature demonstrates great potential in predicting clinical prognosis and drug sensitivity phenotypes in AML patients.

Keywords:  AML; Immunological features; Machine learning; PCD; Prognosis drug response

DOI:  https://doi.org/10.1038/s41598-024-68755-3
STAR Protoc. 2024 Jul 27. pii: S2666-1667(24)00382-4. [Epub ahead of print]5(3): 103217

Protocol for differentiation of monocytes and macrophages from human induced pluripotent stem cells.

Kevin Emmerich, Francesca Calcaterra, Xuming Tang, Guibin Chen, Elena Pontarini, Roberta Ciceri, Dan Yang, Benjamin Joseph, Silvia Della Bella, Isabella Varea, Domenico Mavilio, Manfred Boehm.

  Study of disease-relevant immune cells, namely monocytes and macrophages, is limited based on availability of primary tissue, a limitation that can be remedied using human induced pluripotent stem cell (hiPSC) technology. Here, we present a protocol for differentiation of monocytes and macrophages from hiPSCs. We describe steps for hiPSC maintenance, mesoderm lineage induction, hematopoietic progenitor cells (HPCs) commitment and expansion, and myeloid lineage induction. We then detail procedures for monocyte formation and functional macrophage formation and polarization. For complete details on the use and execution of this protocol, please refer to Chen et al.1.

Keywords:  Cell Biology; Cell Differentiation; Flow Cytometry; Immunology; Microscopy; Stem Cells

DOI:  https://doi.org/10.1016/j.xpro.2024.103217
ACS Nano. 2024 Jul 31.

Biomineralization-Tuned Nanounits Reprogram the Signal Transducer and Activator of Transcription 3 Signaling for Ferroptosis-Immunotherapy in Cancer Stem Cells.

Youbo Zhao, Yang Fei, Yang Zhao, Menghuan Li, Yan Hu, Kaiyong Cai, Shu-Hong Yu, Zhong Luo.

  Cancer stem cells (CSCs) are promising targets for improving anticancer treatment outcomes while eliminating recurrence, but their treatment remains a major challenge. Here, we report a nanointegrative strategy to realize CSC-targeted ferroptosis-immunotherapy through spatiotemporally controlled reprogramming of STAT3-regulated signaling circuits. Specifically, STAT3 inhibitor niclosamide (Ni) and an experimental ferroptosis drug (1S, 3R)-RSL3 (RSL3) are integrated into hyaluronic acid-modified amorphous calcium phosphate (ACP) nanounits through biomineralization (CaP-PEG-HA@Ni/RSL3), which could be recognized by CD44-overexpressing CSCs and released in a synchronized manner. Ni inhibits the CSC-intrinsic STAT3-PD-L1 axis to stimulate adaptive immunity and enhance interferon gamma (IFNγ) secretion by CD8+ T cells to downregulate SLC7A11 and SLC3A2 for blocking glutathione biosynthesis. Meanwhile, Ni-dependent STAT3 inhibition also upregulates ACSL4 through downstream signaling and IFNγ feedback. These effects cooperate with RSL3-mediated GPX4 deactivation to induce pronounced ferroptosis. Furthermore, CaP-PEG-HA@Ni/RSL3 also impairs the immunosuppressive M2-like tumor-associated macrophages, while Ca2+ ions released from degraded ACP could chelate with lipid peroxides in ferroptotic CSCs to avoid CD8+ T-cell inhibition, thus boosting the effector function of activated CD8+ T cells. This study offers a cooperative ferroptosis-immunotherapeutic approach for the treatment of refractory cancer.

Keywords:  STAT3; biomineralized nanoassemblies; cancer stem cell treatment; ferroptosis; immunotherapy

DOI:  https://doi.org/10.1021/acsnano.4c05084
Sci Adv. 2024 Aug 02. 10(31): eadj3145

Caspase-2 is essential for proliferation and self-renewal of nucleophosmin-mutated acute myeloid leukemia.

Dharaniya Sakthivel, Alexandra N Brown-Suedel, Karla E Lopez, Suruchi Salgar, Luiza E Coutinho, Francesca Keane, Shixia Huang, Kenneth Mc Sherry, Chloé I Charendoff, Kevin P Dunne, Dexter J Robichaux, Alexander Vargas-Hernández, BaoChau Le, Crystal S Shin, Alexandre F Carisey, Marcin Poreba, Jonathan M Flanagan, Lisa Bouchier-Hayes.

Mutation in nucleophosmin (NPM1) causes relocalization of this normally nucleolar protein to the cytoplasm (NPM1c+). Despite NPM1 mutation being the most common driver mutation in cytogenetically normal adult acute myeloid leukemia (AML), the mechanisms of NPM1c+-induced leukemogenesis remain unclear. Caspase-2 is a proapoptotic protein activated by NPM1 in the nucleolus. Here, we show that caspase-2 is also activated by NPM1c+ in the cytoplasm and DNA damage-induced apoptosis is caspase-2 dependent in NPM1c+ but not in NPM1wt AML cells. Strikingly, in NPM1c+ cells, caspase-2 loss results in profound cell cycle arrest, differentiation, and down-regulation of stem cell pathways that regulate pluripotency including impairment of the AKT/mTORC1 pathways, and inhibition of Rictor cleavage. In contrast, there were minimal differences in proliferation, differentiation, or the transcriptional profile of NPM1wt cells lacking caspase-2. Our results show that caspase-2 is essential for proliferation and self-renewal of AML cells expressing mutated NPM1. This study demonstrates that caspase-2 is a major effector of NPM1c+ function.

DOI: https://doi.org/10.1126/sciadv.adj3145
Front Immunol. 2024 ;15 1409945

Immune infiltration-related genes regulate the progression of AML by invading the bone marrow microenvironment.

Shuangmei Yu, Jiquan Jiang.

  In this study, we try to find the pathogenic role of immune-related genes in the bone marrow microenvironment of AML. Through WGCNA, seven modules were obtained, among which the turquoise module containing 1793 genes was highly correlated with the immune infiltration score. By unsupervised clustering, the turquoise module was divided into two clusters: the intersection of clinically significant genes in the TCGA and DEGs to obtain 178 genes for mutation analysis, followed by obtaining 17 genes with high mutation frequency. Subsequently, these 17 genes were subjected to LASSO regression analysis to construct a riskscore model of 8 hub genes. The TIMER database, ImmuCellAI portal website, and ssGSEA elucidate that the hub genes and risk scores are closely related to immune cell infiltration into the bone marrow microenvironment. In addition, we also validated the relative expression levels of hub genes using the TCGA database and GSE114868, and additional expression levels of hub genes in AML cell lines in vitro. Therefore, we constructed an immune infiltration-related gene model that identify 8 hub genes with good risk stratification and predictive prognosis for AML.

Keywords:  LASSO; WGCNA; acute myeloid leukemia; bone marrow microenvironment; immune infiltration analysis; immune infiltration-related genes (IIRGs)

DOI:  https://doi.org/10.3389/fimmu.2024.1409945
Biomol Ther (Seoul). 2024 Aug 02.

Gilteritinib Reduces FLT3 Expression in Acute Myeloid Leukemia Cells.

Thị Lam Thái, Sun-Young Han.

  Acute myeloid leukemia (AML) is a genetically diverse and challenging malignancy, with mutations in the FLT3 gene being particularly common and deleterious. Gilteritinib, a potent FLT3 inhibitor, has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of relapsed/refractory AML with FLT3 mutations. Although gilteritinib was developed based on its inhibitory activity against FLT3 kinase, it is important to understand the precise mechanisms of its antileukemic activity in managing drug resistance and discovering biomarkers. This study was designed to elucidate the effect of gilteritinib on the FLT3 expression level. The results showed that gilteritinib induced a dose-dependent decrease in both FLT3 phosphorylation and expression. This reduction was particularly pronounced after 48 h of treatment. The decrease in FLT3 expression was found to be independent of changes in FLT3 mRNA transcription, suggesting post-transcriptional regulatory mechanisms. Further studies were performed in various AML cell lines and cells with both FLT3 wild-type and FLT3 mutant exhibited FLT3 reduction by gilteritinib treatment. In addition, other FLT3 inhibitors were evaluated for their ability to reduce FLT3 expression. Other FLT3 inhibitors, midostaurin, crenolanib, and quizartinib, also reduced FLT3 expression, consistent with the effect of gilteritinib. These findings hold great promise for optimizing gilteritinib treatment in AML patients. However, it is important to recognize that further research is warranted to gain a full understanding of these mechanisms and their clinical implications in the context of FLT3 reduction.

Keywords:  Acute myeloid leukemia; Antileukemic activity; FLT3; FLT3 expression; Gilteritinib; Kinase inhibitors

DOI:  https://doi.org/10.4062/biomolther.2023.215