bims-scepro 2025-01-19 papers

bims-scepro

Biomed News

on Stem cell proteostasis

Issue of 2025–01–19
25 papers selected by
William Grey, University of York

Integrated Local and Systemic Communication Factors Regulate Nascent Hematopoietic Progenitor Escape During Developmental Hematopoiesis.
RSK1 is an exploitable dependency in myeloproliferative neoplasms and secondary acute myeloid leukemia.
Protocol for differentiating hematopoietic progenitor cells from human pluripotent stem cells in chemically defined monolayer culture.
CEBPA repression by MECOM blocks differentiation to drive aggressive leukemias.
A Novel In Vitro Model of the Bone Marrow Microenvironment in Acute Myeloid Leukemia Identifies CD44 and Focal Adhesion Kinase as Therapeutic Targets to Reverse Cell Adhesion-Mediated Drug Resistance.
U2AF1 mutation causes an oxidative stress and DNA repair defect in hematopoietic and leukemic cells.
Protocol for isolation of human bone marrow stromal cells and characterization of cellular metabolism.
Monocytes generated by interleukin-6-treated human hematopoietic stem and progenitor cells secrete calprotectin that inhibits erythropoiesis.
Challenging the Astral mass analyzer to quantify up to 5,300 proteins per single cell at unseen accuracy to uncover cellular heterogeneity.
CPSF6-RARγ interacts with histone deacetylase 3 to promote myeloid transformation in RARG-fusion acute myeloid leukemia.
Targeting deubiquitinase USP7-mediated stabilization of XPO1 contributes to the anti-myeloma effects of selinexor.
Inducing apoptosis in acute myeloid leukemia; mechanisms and limitations.
Targeting CD45 by gene-edited CAR T cells for leukemia eradication and hematopoietic stem cell transplantation preconditioning.
Loss of the Inv(16) Oncogene CBFB::MYH11 Eliminates Leukemia from the Blood and Spleen, but not the Bone Marrow.
Enhanced kinase translocation reporters for simultaneous real-time measurement of PKA, ERK, and calcium.
iPSC stem cell banks: is it really necessary if we already have cord blood banks?
Restoring hematopoietic stem and progenitor cell function in Fancc -/- mice by in situ delivery of RNA lipid nanoparticles.
NVP-2, in combination with Orlistat, represents a promising therapeutic strategy for acute myeloid leukemia.
Diverse microtubule-binding repeats regulate TPX2 activities at distinct locations within the spindle.
mTOR signalling controls protein aggregation during heat stress and cellular aging in a translation- and Hsf1-independent manner.
FT538, iPSC-derived NK cells, enhance AML cell killing when combined with chemotherapy.
Proteasome inhibition induces microtubule-dependent changes in nuclear morphology.
SYS-1/beta-catenin inheritance and regulation by Wnt-signaling during asymmetric cell division.
Activation of the 26S Proteasome to Reduce Proteotoxic Stress and Improve the Efficacy of PROTACs.
Combined anti-leukemic effect of gilteritinib and GSK-J4 in FLT3-ITD+ acute myeloid leukemia.

Int J Mol Sci. 2024 Dec 31. pii: 301. [Epub ahead of print]26(1):

Integrated Local and Systemic Communication Factors Regulate Nascent Hematopoietic Progenitor Escape During Developmental Hematopoiesis.

Carson Shalaby, James Garifallou, Christopher S Thom.

  Mammalian blood cells originate from specialized 'hemogenic' endothelial (HE) cells in major arteries. During the endothelial-to-hematopoietic transition (EHT), nascent hematopoietic stem cells (HSCs) bud from the arterial endothelial wall and enter circulation, destined to colonize the fetal liver before ultimately migrating to the bone marrow. Mechanisms and processes that facilitate EHT and the release of nascent HSCs are incompletely understood, but may involve signaling from neighboring vascular endothelial cells, stromal support cells, circulating pre-formed hematopoietic cells, and/or systemic factors secreted by distal organs. We used single cell RNA sequencing analysis from human embryonic cells to identify relevant signaling pathways that support nascent HSC release. In addition to intercellular and secreted signaling modalities that have been previously functionally validated to support EHT and/or developmental hematopoiesis in model systems, we identify several novel modalities with plausible mechanisms to support EHT and HSC release. Our findings paint a portrait of the complex inter-regulated signals from the local niche, circulating hematopoietic/inflammatory cells, and distal fetal liver that support hematopoiesis.

Keywords:  blood; endothelium; hematopoiesis; signaling; stroma

DOI:  https://doi.org/10.3390/ijms26010301
Nat Commun. 2025 Jan 16. 16(1): 492

RSK1 is an exploitable dependency in myeloproliferative neoplasms and secondary acute myeloid leukemia.

Tim Kong, Angelo B A Laranjeira, Christopher T Letson, LaYow Yu, Shuyang Lin, Jared S Fowles, Daniel A C Fisher, Sherwin Ng, Wei Yang, Fan He, Minyoung Youn, Kailen Mark, Ana San Jose, Jingxian Liu, Alexander B Kim, Maggie J Cox, Mary C Fulbright, Aarthi Jayanthan, Gerrit Los, Stacey L Rentschler, Li Ding, Kathleen M Sakamoto, Sandra E Dunn, Grant A Challen, Stephen T Oh.

Myeloid malignancies are heterogenous disorders characterized by distinct molecular drivers but share convergence of oncogenic signaling pathways and propagation by ripe pro-inflammatory niches. Here, we establish a comprehensive transcriptional atlas across the spectrum of myeloproliferative neoplasms (MPN) and secondary acute myeloid leukemia (sAML) through RNA-sequencing of 158 primary samples encompassing CD34+ hematopoietic stem/progenitor cells and CD14+ monocytes. Supported by mass cytometry (CyTOF) profiling, we reveal aberrant networks of PI3K/AKT/mTOR signalling and NFκB-mediated hyper-inflammation. Combining ATAC-Seq, CUT&Tag, RNA-seq, and CyTOF, we demonstrate that targeting of ribosomal protein S6 kinase A1 (RSK1) suppresses NFκB activation and diminishes pro-inflammatory mediators including tumor necrosis factor (TNF) associated with MPN disease severity and transformation. We further evaluate a therapeutic approach utilizing a first-in-class RSK inhibitor, PMD-026, currently in Phase 2 development for breast cancer, for use in myeloid malignancies. Treatment with PMD-026 suppressed disease burden across seven syngeneic and patient-derived xenograft leukemia mouse models spanning the spectrum of driver and disease-modifying mutations. These findings uncover a therapeutic avenue for a conserved dependency across MPN and sAML.

DOI: https://doi.org/10.1038/s41467-024-55643-7
STAR Protoc. 2025 Jan 11. pii: S2666-1667(24)00710-X. [Epub ahead of print]6(1): 103545

Protocol for differentiating hematopoietic progenitor cells from human pluripotent stem cells in chemically defined monolayer culture.

Shaokang Mo, Kengyuan Qu, Jun Shen, Kuangyu Yen.

  Human pluripotent stem cells (hPSCs) provide a powerful platform for generating hematopoietic progenitor cells (HPCs) and investigating hematopoietic development. Here, we present a protocol for maintaining hPSCs and inducing their differentiation into HPCs through the endothelial-to-hematopoietic transition (EHT) on vitronectin-coated plates. We outline steps for evaluating the efficiency of HPC generation and assessing their potential to differentiate into various hematopoietic lineages. This protocol serves as a framework for exploring human hematopoiesis and generating various functional blood cells. For complete details on the use and execution of this protocol, please refer to Shen et al.1 and Qu et al.2.

Keywords:  cell biology; cell culture; cell differentiation; developmental biology; stem cells

DOI:  https://doi.org/10.1016/j.xpro.2024.103545
bioRxiv. 2024 Dec 30. pii: 2024.12.30.630680. [Epub ahead of print]

CEBPA repression by MECOM blocks differentiation to drive aggressive leukemias.

Travis J Fleming, Mateusz Antoszewski, Sander Lambo, Michael C Gundry, Riccardo Piussi, Lara Wahlster, Sanjana Shah, Fiona E Reed, Kevin D Dong, Joao A Paulo, Steven P Gygi, Claudia Mimoso, Seth R Goldman, Karen Adelman, Jennifer A Perry, Yana Pikman, Kimberly Stegmaier, Maria N Barrachina, Kellie R Machlus, Volker Hovestadt, Andrea Arruda, Mark D Minden, Richard A Voit, Vijay G Sankaran.

Acute myeloid leukemias (AMLs) have an overall poor prognosis with many high-risk cases co-opting stem cell gene regulatory programs, yet the mechanisms through which this occurs remain poorly understood. Increased expression of the stem cell transcription factor, MECOM, underlies one key driver mechanism in largely incurable AMLs. How MECOM results in such aggressive AML phenotypes remains unknown. To address existing experimental limitations, we engineered and applied targeted protein degradation with functional genomic readouts to demonstrate that MECOM promotes malignant stem cell-like states by directly repressing pro-differentiation gene regulatory programs. Remarkably and unexpectedly, a single node in this network, a MECOM-bound cis-regulatory element located 42 kb downstream of the myeloid differentiation regulator CEBPA, is both necessary and sufficient for maintaining MECOM-driven leukemias. Importantly, targeted activation of this regulatory element promotes differentiation of these aggressive AMLs and reduces leukemia burden in vivo, suggesting a broadly applicable differentiation-based approach for improving therapy.

DOI: https://doi.org/10.1101/2024.12.30.630680
Cancers (Basel). 2025 Jan 03. pii: 135. [Epub ahead of print]17(1):

A Novel In Vitro Model of the Bone Marrow Microenvironment in Acute Myeloid Leukemia Identifies CD44 and Focal Adhesion Kinase as Therapeutic Targets to Reverse Cell Adhesion-Mediated Drug Resistance.

Eleni E Ladikou, Kim Sharp, Fabio A Simoes, John R Jones, Thomas Burley, Lauren Stott, Aimilia Vareli, Emma Kennedy, Sophie Vause, Timothy Chevassut, Amarpreet Devi, Iona Ashworth, David M Ross, Tanja Nicole Hartmann, Simon A Mitchell, Chris J Pepper, Giles Best, Andrea G S Pepper.

   BACKGROUND/OBJECTIVES: Acute myeloid leukemia (AML) is an aggressive neoplasm. Although most patients respond to induction therapy, they commonly relapse due to recurrent disease in the bone marrow microenvironment (BMME). So, the disruption of the BMME, releasing tumor cells into the peripheral circulation, has therapeutic potential.
METHODS: Using both primary donor AML cells and cell lines, we developed an in vitro co-culture model of the AML BMME. We used this model to identify the most effective agent(s) to block AML cell adherence and reverse adhesion-mediated treatment resistance.
RESULTS: We identified that anti-CD44 treatment significantly increased the efficacy of cytarabine. However, some AML cells remained adhered, and transcriptional analysis identified focal adhesion kinase (FAK) signaling as a contributing factor; the adhered cells showed elevated FAK phosphorylation that was reduced by the FAK inhibitor, defactinib. Importantly, we demonstrated that anti-CD44 and defactinib were highly synergistic at diminishing the adhesion of the most primitive CD34high AML cells in primary autologous co-cultures.
CONCLUSIONS: Taken together, we identified anti-CD44 and defactinib as a promising therapeutic combination to release AML cells from the chemoprotective AML BMME. As anti-CD44 is already available as a recombinant humanized monoclonal antibody, the combination of this agent with defactinib could be rapidly tested in AML clinical trials.

Keywords:  CAM-DR; CD44; FAK; acute myeloid leukemia (AML); adhesion; bone marrow microenvironment

DOI:  https://doi.org/10.3390/cancers17010135
Free Radic Biol Med. 2025 Jan 13. pii: S0891-5849(25)00019-X. [Epub ahead of print]

U2AF1 mutation causes an oxidative stress and DNA repair defect in hematopoietic and leukemic cells.

Yishuang Li, Xuanjia Dong, Haiyan Xing, Wenbing Liu, Runxia Gu, Shaowei Qiu, Yingxi Xu, Hui Wei, Min Wang, Guoguang Zheng, Qing Rao, Jianxiang Wang.

  U2AF1 is a core component of spliceosome and controls cell-fate specific alternative splicing. U2AF1 mutations have been frequently identified in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) patients, and mutations in U2AF1 are associated with poor prognosis in hematopoietic malignant diseases. Here, by forced expression of mutant U2AF1 (U2AF1 S34F) in hematopoietic and leukemic cell lines, we find that U2AF1 S34F causes increased reactive oxygen species (ROS) production. In hematopoietic cell line, a defect in mitochondrial function and DNA damage response deficiency are found in U2AF1 S34F expressing 32D cells. In leukemic cell line Molm13 cells, U2AF1 mutation leads to resistance to DNA damaging agents. Accumulation of DNA damage is also found in U2AF1 S34F expressing leukemic cells when treated with DNA damage agent. Finally, in our established hematopoietic-specific U2af1 S34F knock-in mice model, U2AF1 mutation leads to the development of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) and causes DNA damage accumulation in hematopoietic cells. Our study provides evidence that U2AF1 mutation causes DNA damage response deficiency and DNA damage accumulation in hematopoietic cells, and suggests that mutant U2AF1 induced higher ROS production, resistance to DNA damaging agents and increased genomic instability may contribute to poor prognosis of AML patients with U2AF1 mutations.

Keywords:  DNA damage accumulation,hematopoietic cell; DNA repair response; U2AF1 mutation; oxidative stress

DOI:  https://doi.org/10.1016/j.freeradbiomed.2025.01.019
STAR Protoc. 2025 Jan 13. pii: S2666-1667(24)00718-4. [Epub ahead of print]6(1): 103553

Protocol for isolation of human bone marrow stromal cells and characterization of cellular metabolism.

Martina Dzubanova, Michaela Ferencakova, Andrea Benova, Dalia Ali, Michaela Tencerova.

  Bone marrow stromal cells (BMSCs) serve as a valuable reservoir of multipotent stem cells important in the regulation of bone homeostasis and energy metabolism. Here, we present a protocol for isolating human BMSCs (hBMSCs) and characterizing their cellular metabolism related to hBMSC functional properties. We describe steps for bioenergetics, cell senescence, and production of reactive oxygen species (ROS), together with description of the data analysis. These assays provide information on hBMSC metabolic status valuable to regenerative medicine and therapeutic applications. For complete details on the use and execution of this protocol, please refer to Tencerova et al.1.

Keywords:  Cell Biology; Metabolism; Stem Cells

DOI:  https://doi.org/10.1016/j.xpro.2024.103553
iScience. 2025 Jan 17. 28(1): 111522

Monocytes generated by interleukin-6-treated human hematopoietic stem and progenitor cells secrete calprotectin that inhibits erythropoiesis.

Valentine Marchand, Lucie Laplane, Louis Valensi, Isabelle Plo, Marine Aglave, Aymeric Silvin, Florence Pasquier, Françoise Porteu, William Vainchenker, Dorothée Selimoglu-Buet, Nathalie Droin, Hana Raslova, Virginie Marcel, Jean-Jacques Diaz, Michaela Fontenay, Eric Solary.

  Elevated circulating levels of calprotectin (CAL), the S100A8/A9 heterodimer, are biomarkers of severe systemic inflammation. Here, we investigate the effects of CAL on early human hematopoiesis. CAL demonstrates limited impact on gene expression in stem and progenitor cells, in contrast with interleukin-6 (IL6), which promotes the expression of the S100A8 and S100A9 genes in hematopoietic progenitors and the generation of monocytes that release CAL. The main target of CAL is an erythroid-megakaryocyte progenitor (EMP) subset. CAL prevents both erythropoietin-driven differentiation of healthy progenitors and JAK2-V617F-driven erythropoiesis. In the context of JAK2-V617F, CAL also promotes the expression of S100A8 and S100A9 genes in monocytes. The signature of CAL effects is detected in the bone marrow progenitors of patients with myeloid malignancy or severe infection. These results position CAL as a mediator of IL6 effects on triggering anemia during inflammation, an effect that is amplified in the context of JAK2-V617F-driven hematopoiesis.

Keywords:  Cell biology; Pathology

DOI:  https://doi.org/10.1016/j.isci.2024.111522
Nat Methods. 2025 Jan 16.

Challenging the Astral mass analyzer to quantify up to 5,300 proteins per single cell at unseen accuracy to uncover cellular heterogeneity.

Julia A Bubis, Tabiwang N Arrey, Eugen Damoc, Bernard Delanghe, Jana Slovakova, Theresa M Sommer, Harunobu Kagawa, Peter Pichler, Nicolas Rivron, Karl Mechtler, Manuel Matzinger.

Despite significant advancements in sample preparation, instrumentation and data analysis, single-cell proteomics is currently limited by proteomic depth and quantitative performance. Here we demonstrate highly improved depth of proteome coverage as well as accuracy and precision for quantification of ultra-low input amounts. Using a tailored library, we identify up to 7,400 protein groups from as little as 250 pg of HeLa cell peptides at a throughput of 50 samples per day. Using a two-proteome mix, we check for optimal parameters of quantification and show that fold change differences of 2 can still be successfully determined at single-cell-level inputs. Eventually, we apply our workflow to A549 cells, yielding a proteome coverage ranging from 1,801 to a maximum of >5,300 protein groups from a single cell depending on cell size and search strategy used, which allows for the study of dependencies between cell size and cell cycle phase. Additionally, our workflow enables us to distinguish between in vitro analogs of two human blastocyst lineages: naive human pluripotent stem cells (epiblast) and trophectoderm-like cells. Our data harmoniously align with transcriptomic data, indicating that single-cell proteomics possesses the capability to identify biologically relevant differences within the blastocyst.

DOI: https://doi.org/10.1038/s41592-024-02559-1
Nat Commun. 2025 Jan 13. 16(1): 616

CPSF6-RARγ interacts with histone deacetylase 3 to promote myeloid transformation in RARG-fusion acute myeloid leukemia.

Tianhui Liu, Tanzhen Wang, Lijuan Qi, Yujie Liu, Meng Shan, Fuqiang Wang, Yanglan Fang, Sining Liu, Lijun Wen, Suning Chen, Depei Wu, Yang Xu.

Acute myeloid leukemia (AML) with retinoic acid receptor gamma (RARG) fusions, which exhibits clinical features resembling acute promyelocytic leukemia (APL), has been identified as a new subtype with poor clinical outcomes. The underlying mechanism of RARG-fusion leukemia remains poorly understood, and needs to be explored urgently to instruct developing effective therapeutic strategies. Here, using the most prevalent RARG fusion, CPSF6-RARG (CR), as a representative, we reveal that the CR fusion, enhances the expansion of myeloid progenitors, impairs their maturation and synergizes with RAS mutations to drive more aggressive myeloid malignancies. Mechanistically, CR fusion interacts with histone deacetylase 3 (HDAC3) to suppress expression of genes associated with myeloid differentiation including the myeloid transcription factor PU.1. Disrupting CR-HDAC3 interaction, restores PU.1 expression and myeloid differentiation. Furthermore, HDAC inhibitors effectively suppress CR-driven leukemia in vitro and in vivo. Hence, our data reveals the molecular bases of oncogenic CR fusion and provides a potential therapeutic approach against AML with CR fusion.

DOI: https://doi.org/10.1038/s41467-024-54860-4
J Transl Med. 2025 Jan 13. 23(1): 62

Targeting deubiquitinase USP7-mediated stabilization of XPO1 contributes to the anti-myeloma effects of selinexor.

Junying Wang, Mengping Chen, Jinxing Jiang, Yike Wan, Xin Li, Minyue Zhang, Fei Xiao, Lu Zhong, Hua Zhong, Zhaoyu Qin, Jian Hou.

   BACKGROUND: Targeting exportin1 (XPO1) with Selinexor (SEL) is a promising therapeutic strategy for patients with multiple myeloma (MM). However, intrinsic and acquired drug resistance constitute great challenges. SEL has been reported to promote the degradation of XPO1 protein in tumor cells. Nevertheless, in myeloma, the precise mechanisms underlying SEL-induced XPO1 degradation and its impact on drug responsiveness remain largely undefined.
METHODS: We assessed XPO1 protein and mRNA levels using western blotting and RT-qPCR. Cycloheximide (CHX) chase assays and degradation blockade assays were used to determine the pathway of XPO1 degradation induced by SEL. The sensitivity of MM cell lines to SEL was evaluated using CCK8-based cell viability assays and AV-PI staining-based cell apoptosis assays. The subcellular localization of the cargo protein RanBP1 was assessed via immunofluorescence staining. Immunoprecipitation coupled with mass spectrometry (IP-MS), bioinformatics analysis and ubiquitination assays, were employed to identify the molecular targets responsible for SEL-induced degradation of XPO1. shRNA-mediated knockdown assays and small molecule inhibitors of USP7 were utilized to disrupt the function of USP7. The role of USP7 in modulating SEL sensitivity was analyzed in MM cell lines, primary CD138+ cells, and xenograft mouse models.
RESULTS: SEL promotes the degradation of XPO1 in MM cells through the ubiquitin-proteasome pathway. There is a positive correlation between XPO1 degradation and sensitivity to SEL in these cells. Inhibiting XPO1 degradation reduces the functional inhibitory effects of SEL on XPO1, as evidenced by decreased nuclear localization of the cargo protein RanBP1. USP7 stabilizes XPO1 in MM cells via its deubiquitinating activity. SEL accelerates the ubiquitination and subsequent degradation of XPO1 by disrupting the interaction between XPO1 and USP7. The expression of USP7 is negatively correlated with patient prognosis and the sensitivity of MM cells to SEL. Inactivating or knocking down USP7 significantly enhances the anti-myeloma effects of SEL both in vitro and in vivo.
CONCLUSION: In conclusion, our findings underscore the essential role of XPO1 degradation in the anti-myeloma efficacy of SEL and establish a research foundation for targeting USP7 to improve the effectiveness of SEL-based therapies in MM.

Keywords:  Drug sensitivity; Multiple myeloma; Protein degradation; Selinexor; USP7; XPO1

DOI:  https://doi.org/10.1186/s12967-025-06068-3
Heliyon. 2025 Jan 15. 11(1): e41355

Inducing apoptosis in acute myeloid leukemia; mechanisms and limitations.

Zahra Koolivand, Farbod Bahreini, Elham Rayzan, Nima Rezaei.

  Acute myeloid leukemia is the expansion of leukemic stem cells which might originate from a stem cell or a progenitor which has acquired self-renewal capacity. An aggregation of leukemic blasts in bone marrow, peripheral blood, and extramedullary tissue will result in acute myeloid leukemia. The main difficulty in treating acute myeloid leukemia is multidrug resistance, leading to treatment failure. This unfortunate phenomenon is practically elevated because of apoptosis inhibition in tumor cells. Two general apoptotic pathways are the Bcl-2 regulated pathway (the intrinsic pathway) and the death receptor pathway. Deficiencies in each of these apoptotic pathways can cause the usual resistance mechanism in this disease. This article reviews and highlights different antiapoptotic pathways, currently-used treatments, and new findings in this field, which may lead to the development of treatment methods for acute myeloid leukemia.

Keywords:  Acute myeloid leukemia; Apoptosis; BCL2 protein; Multiple drug resistance; Treatment failure

DOI:  https://doi.org/10.1016/j.heliyon.2024.e41355
Mol Ther Oncol. 2024 Sep 19. 32(3): 200843

Targeting CD45 by gene-edited CAR T cells for leukemia eradication and hematopoietic stem cell transplantation preconditioning.

Valeria M Stepanova, Dmitry V Volkov, Daria S Osipova, Wenjian Wang, Yingqin Hou, Dmitry E Pershin, Mariia S Fadeeva, Ekaterina A Malakhova, Elena A Kulakovskaya, Lui Cuicui, Zhao Mingfeng, Hongkai Zhang, Jia Xie, Ding Zhang, Ilgar Z Mamedov, Alexandr S Chernov, Georgij B Telegin, Yuri P Rubtsov, Alexander G Gabibov, Peng Wu, Michael A Maschan, Alexey V Stepanov.

  Hematopoietic stem cell transplantation (HSCT) is widely used to treat patients with life-threatening hematologic and immune system disorders. Current nontargeted chemo-/radiotherapy conditioning regimens cause tissue injury and induce an array of immediate and delayed adverse effects, limiting the application of this life-saving treatment. The growing demand to replace canonical conditioning regimens has led to the development of alternative approaches, such as antibody-drug conjugates, naked antibodies, and CAR T cells. Here, we introduce a preconditioning strategy targeting CD45 on hematopoietic cells with CAR45 T cells. To avoid fratricide of CD45 CAR T cells, genomic disruption of the CD45 gene was performed on human CD45 CAR T cells in combination with the signaling kinase inhibitor dasatinib. CD45Δ CAR45 T cells showed high cytotoxicity in vitro and depletion of tumor cells in vivo. These cells were effective in elimination of human hematopoietic cells engrafted in humanized immunodeficient mice by transfusion with human blood-derived hematopoietic stem cells (HSCs). Similarly, CD45Δ CAR45 natural killer (NK) cells exhibited potent cytotoxicity toward tumor cell lines and human hematopoietic cells in vitro. Thus, we provide the proof of concept for the generation and preclinical efficacy of fratricide-resistant CAR45 T and NK cells directed against CD45-expressing tumors and hematopoietic cells.

Keywords:  CD45 knockout; HSCT preconditioning; fratricide-resistant CAR45 NK; fratricide-resistant CAR45 T; leukemia

DOI:  https://doi.org/10.1016/j.omton.2024.200843
bioRxiv. 2024 Oct 06. pii: 2024.10.06.616896. [Epub ahead of print]

Loss of the Inv(16) Oncogene CBFB::MYH11 Eliminates Leukemia from the Blood and Spleen, but not the Bone Marrow.

Sipra Panda, Yiqian Wang, Michelle Becker, Arjun Dhir, Calvin Lam, Cecilia Rivas, Lemlem Alemu, Lisa Garrett, Samantha Swenson, R Katherine Hyde.

Inversion of chromosome 16 [inv(16)] is one of the most common chromosomal rearrangements in Acute Myeloid Leukemia (AML) and generates the fusion gene CBFB::MYH11 (CM) , which initiates leukemogenesis. Patients with inv(16) at diagnosis invariably have the rearrangement at relapse, leading to the assumption that CM is required after leukemic transformation. However, this has yet to be shown experimentally. Using a knock-in mouse that allows for deletion of CM after leukemia development, we found that loss of the fusion gene increased apoptosis and decreased colony growth in vitro . Interestingly, 5-20% of the colonies had successfully deleted CM . To test the role of CM in vivo , we used an inducible shRNA knockdown (KD) construct against the fusion gene. We found that decreased CM expression eliminated leukemia cells from the peripheral blood and spleen, but not the bone marrow, despite these cells showing significant knockdown of CM at the mRNA and protein levels. Furthermore, with prolonged KD of CM , ∼40% of mice re-established disease while maintaining KD of the fusion gene. Our work indicates that CM is required by leukemia cells in the spleen and blood, but that cells in the bone marrow can survive and re-establish disease independent of the fusion protein.

DOI: https://doi.org/10.1101/2024.10.06.616896
J Biol Chem. 2025 Jan 13. pii: S0021-9258(25)00030-4. [Epub ahead of print] 108183

Enhanced kinase translocation reporters for simultaneous real-time measurement of PKA, ERK, and calcium.

Shang-Jui Tsai, Yijing Gong, Austin Dabbs, Fiddia Zahra, Junhao Xu, Aleksander Geske, Michael J Caterina, Stephen J Gould.

Kinase translocation reporters (KTRs) are powerful tools for single-cell measurement of time-integrated kinase activity but suffer from restricted dynamic range and limited sensitivity, particularly in neurons. To address these limitations, we developed enhanced KTRs (eKTRs) for protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) by (i) increasing KTR size, which reduces the confounding effect of KTR diffusion through the nuclear pore, and (ii) modulating the strength of the bipartite nuclear localization signal (bNLS) in their kinase sensor domains, to ensures that the relative distribution of the KTR between the nucleus and cytoplasmic is determined by active nuclear import, active nuclear export, and relative activity of their cognate kinase. The resultant sets of ePKA-KTRs and eERK-KTRs display high sensitivity, broad dynamic range, and cell type-specific tuning. Moreover, co-expression of optically separable ePKA-KTRs and eERK-KTRs allowed us to simultaneously monitor the activation and inhibition of PKA and ERK in live cells, which verified that these eKTRs respond as expected to direct agonists and inhibitors, and that crosstalk between these pathways is highly unbalanced, with activation of PKA suppressing ERK activity, while activation of ERK induces PKA activity. Taken together, our findings highlight the importance of KTR size and bNLS strength to KTR sensitivity and dynamic range, show that different cell types require different eKTRs, and identify ePKA-KTR1.4 and eERK-KTR1.2 as particularly well-suited for monitoring PKA and ERK in primary sensory neurons.

DOI: https://doi.org/10.1016/j.jbc.2025.108183
Regen Med. 2025 Jan 16. 1-4

iPSC stem cell banks: is it really necessary if we already have cord blood banks?

David T Harris, Michael Badowski.



Keywords:  HLA matching; Stem cells; cord blood; iPS cells; regenerative medicine; transplant

DOI:  https://doi.org/10.1080/17460751.2025.2453332
Mol Ther Nucleic Acids. 2025 Mar 11. 36(1): 102423

Restoring hematopoietic stem and progenitor cell function in Fancc -/- mice by in situ delivery of RNA lipid nanoparticles.

Omar Banda, Sarah E Adams, Linah Omer, Seul K Jung, Hooda Said, Theerapat Phoka, Ying Tam, Drew Weissman, Stefano Rivella, Mohamad-Gabriel Alameh, Peter Kurre.

  Fanconi anemia (FA) is a congenital multisystem disorder characterized by early-onset bone marrow failure (BMF) and cancer susceptibility. While ex vivo gene addition and repair therapies are being considered as treatment options, depleted hematopoietic stem cell (HSC) pools, poor HSC mobilization, compromised survival during ex vivo transduction, and increased sensitivity to conventional conditioning strategies limit eligibility for FA patients to receive gene therapies. As an alternative approach, we explored in vivo protein replacement by mRNA delivery via lipid nanoparticles (LNPs). Our study aims to address several key obstacles to current mRNA-LNP treatment: access to the HSC niche, effective expression half-life, and potential mRNA LNP immunogenicity. Results demonstrate efficient in vivo LNP transfection of murine BM via intravenous or intrafemoral injections, yielding reporter expression across hematopoietic and non-hematopoietic BM niche populations. Functionally, LNP delivery of modified Fancc mRNA restored ex vivo expansion. In a proof of principle approach, LNP-treated murine Fancc -/- HSPCs engrafted with restored alkylator resistance up to 120 h post-treatment using circularized mRNA constructs. In vitro delivery of mRNA LNPs resulted in modest differences in innate immune target gene expression in both FA and wild-type HSPCs. Our results suggest that mRNA-LNP-based protein replacement therapy holds promise for clinical translation.

Keywords:  FANCC knockout mouse model; Fanconi anemia; MT: Delivery Strategies; intrafemoral delivery; intraosseous delivery; in vivo delivery; lipid nanoparticles; mRNA therapy; monogenic disorders; protein replacement therapy

DOI:  https://doi.org/10.1016/j.omtn.2024.102423
Cancer Biol Ther. 2025 Dec;26(1): 2450859

NVP-2, in combination with Orlistat, represents a promising therapeutic strategy for acute myeloid leukemia.

Qing Zhu, Jia Cheng, Yuqing Gao, Zimu Zhang, Jian Pan, Xin Su, Danhong Fei, Linbo Cai, Juanjuan Yu, Yanling Chen, Wanyan Jiao, Di Wu, Xiaolu Li, Peifang Xiao.

  Cell cycle dysregulation and the corresponding metabolic reprogramming play significant roles in tumor development and progression. CDK9, a kinase that regulates gene transcription and cell cycle, also induces oncogene transcription and abnormal cell cycle in AML cells. The function of CDK9 for gene regulation in AML cells requires further exploration. In this study, we knocked down the CDK9 to investigate its effects on the growth and survival of AML cells. Through RNA-seq analysis, we identified that in U937 cells CDK9 regulates numerous genes involved in proliferation and apoptosis, including mTOR, SREBF1, and Bcl-2. Furthermore, our results demonstrated that both CDK9 and FASN are crucial for the proliferation and survival of Kasumi-1 and U937 cells. Mechanistically, MCL1, c-Myc, and Akt/mTOR/SREBF1 may be critical factors and pathways in the combined therapy of NVP-2 and Orlistat. In summary, our study revealed that CDK9 and FASN are vital for maintaining AML cell survival and proliferation. Treatment with NVP-2 and Orlistat may be a promising clinical candidate for patients with AML.

Keywords:  Acute myeloid leukemia; CDK9; FASN; SREBF1; apoptosis; c-Myc; cell proliferation; molecular targeted therapy

DOI:  https://doi.org/10.1080/15384047.2025.2450859
J Cell Biol. 2025 Mar 03. pii: e202404025. [Epub ahead of print]224(3):

Diverse microtubule-binding repeats regulate TPX2 activities at distinct locations within the spindle.

Zhuobi Liang, Junjie Huang, Yong Wang, Shasha Hua, Kai Jiang.

TPX2 is an elongated molecule containing multiple α-helical repeats. It stabilizes microtubules (MTs), promotes MT nucleation, and is essential for spindle assembly. However, the molecular basis of how TPX2 performs these functions remains elusive. Here, we systematically characterized the MT-binding activities of all TPX2 modules individually and in combinations and investigated their respective contributions both in vitro and in cells. We show that TPX2 contains α-helical repeats with opposite preferences for "extended" and "compacted" tubulin dimer spacing, and their distinct combinations produce divergent outcomes, making TPX2 activity highly robust yet tunable. Importantly, a repeat group at the C terminus, R8-9, is the key determinant of the TPX2 function. It stabilizes MTs by promoting rescues in vitro and is critical in spindle assembly. We propose a model where TPX2 activities are spatially regulated via its diverse MT-binding repeats to accommodate its varied functions in distinct locations within the spindle. Furthermore, we reveal a synergy between TPX2 and HURP in stabilizing spindle MTs.

DOI: https://doi.org/10.1083/jcb.202404025
J Biol Chem. 2025 Jan 09. pii: S0021-9258(25)00019-5. [Epub ahead of print] 108172

mTOR signalling controls protein aggregation during heat stress and cellular aging in a translation- and Hsf1-independent manner.

Arthur Fischbach, Per O Widlund, Xinxin Hao, Thomas Nyström.

  The mTOR (mechanistic target of rapamycin) signaling pathway appears central to the aging process as genetic or pharmacological inhibition of mTOR extends lifespan in most eukaryotes tested. While the regulation of protein synthesis by mTOR has been studied in great detail, its impact on protein misfolding and aggregation during stress and aging is less explored. In this study, we identified the mTOR signaling pathway and the linked SEA complex as central nodes of protein aggregation during heat stress and cellular aging, using Saccharomyces cerevisiae as a model organism. Based on a synthetic genetic array (SGA) screen, we found that reduced mTOR activity, achieved through deletion of TCO89, an mTORC1 subunit, almost completely prevents protein aggregation during heat stress and aging without reducing global translation rates and independently of an Hsf1-dependent stress response. Conversely, increased mTOR activity, achieved through deletion of NPR3, a SEA complex subunit, exacerbates protein aggregation, but not by over-activating translation. In summary, our work demonstrates that mTOR signaling is a central contributor to age-associated and heat shock-induced protein aggregation and that this is unlinked to quantitatively discernable effects on translation and Hsf1.

Keywords:  Aging; mTOR; protein aggregation; proteinopathy; proteostasis; spatial protein quality control

DOI:  https://doi.org/10.1016/j.jbc.2025.108172
J Cell Mol Med. 2025 Jan;29(1): e70169

FT538, iPSC-derived NK cells, enhance AML cell killing when combined with chemotherapy.

Amanda Eckstrom, Anudishi Tyagi, Sajid Mahmood, Lilly Wong, Bahram Valamehr, Adishwar Rao, Akriti Agrawal, Maryam Siddiqui, V Lokesh Battula.

  Induced pluripotent stem cell (iPSC)-derived natural killer (NK) cells offer an opportunity for a standardized, off-the-shelf treatment with the potential to treat a wider population of acute myeloid leukaemia (AML) patients than the current standard of care. FT538 iPSC-NKs express a high-affinity, noncleavable CD16 to maximize antibody dependent cellular cytotoxicity, a CD38 knockout to improve metabolic fitness, and an IL-15/IL-15 receptor fusion preventing the need for cytokine administration, the main source of adverse effects in NK cell-based therapies. Here, we sought to evaluate the potential of FT538 iPSC-NKs as a therapy for AML through their effect on AML cell lines and primary AML cells. We observed that FT538 iPSC-NKs induce effector-to-target cell ratio dependent apoptosis in cell lines and primary AML cells, including cells from high-risk patients. Flow cytometric analysis revealed that FT538 iPSC-NKs induce AML cell death when combined with the AML therapies: cytarabine, venetoclax and gilteritinib. Moreover, cytarabine did not affect FT538 iPSC-NK viability, suggesting that iPSC-derived NK therapies and chemotherapy may be a promising treatment combination. This study provides the basis for further study of iPSC-derived NK cell therapies as a treatment option for high-risk AML patients, particularly those with disease resistant to standard therapies.

Keywords:  AML; NK cell therapy; NK cell‐mediated apoptosis; iPSC‐derived NK cells; immunotherapy

DOI:  https://doi.org/10.1111/jcmm.70169
iScience. 2025 Jan 17. 28(1): 111550

Proteasome inhibition induces microtubule-dependent changes in nuclear morphology.

Sourabh Sengupta, Abdullah Bashar Sami, Jesse C Gatlin, Daniel L Levy.

  Cancers and neurodegenerative disorders are associated with both disrupted proteostasis and altered nuclear morphology. Determining if changes in nuclear morphology contribute to pathology requires an understanding of the underlying mechanisms, which are difficult to elucidate in cells where pleiotropic effects of altering proteostasis might indirectly influence nuclear morphology. To investigate direct effects, we studied nuclei assembled in Xenopus egg extract where potentially confounding effects of transcription, translation, cell cycle progression, and actin dynamics are absent. We report that proteasome inhibition causes acute microtubule-dependent changes in nuclear morphology and stability and altered microtubule dynamics and organization. Proteomic analysis of proteasome-inhibited extracts identified an increased abundance of microtubule nucleator TubGCP6, and TubGCP6 depletion partially rescued nuclear morphology. Key results were confirmed in HeLa cells. We propose that accumulation of TubGCP6 leads to altered microtubule dynamics proximal to the nucleus, producing forces that deform the nucleus and impact nuclear morphology and integrity.

Keywords:  Biological sciences; Cell biology; Functional aspects of cell biology

DOI:  https://doi.org/10.1016/j.isci.2024.111550
Mol Biol Cell. 2025 Jan 15. mbcE24100441

SYS-1/beta-catenin inheritance and regulation by Wnt-signaling during asymmetric cell division.

Maria F Valdes Michel, Bryan T Phillips.

Asymmetric cell division (ACD) allows daughter cells of a polarized mother to acquire different developmental fates. In C. elegans, the Wnt/β-catenin Asymmetry (WβA) pathway regulates many embryonic and larval ACDs; here, a Wnt gradient induces an asymmetric distribution of Wnt signaling components within the dividing mother cell. One terminal nuclear effector of the WβA pathway is the transcriptional activator SYS-1/β-catenin. SYS-1 is sequentially negatively regulated during ACD; first by centrosomal regulation and subsequent proteasomal degradation and second by asymmetric activity of the β-catenin "destruction complex" in one of the two daughter cells, which decreases SYS-1 levels in the absence of WβA signaling. However, the extent to which mother cell SYS-1 influences cell fate decisions of the daughters is unknown. Here, we quantify inherited SYS-1 in the differentiating daughter cells and the role of SYS-1 inheritance in Wnt-directed ACD. Photobleaching experiments demonstrate the GFP::SYS-1 present in daughter cell nuclei is comprised of inherited and de novo translated SYS-1 pools. We used a photoconvertible DENDRA2::SYS-1, to directly observe the dynamics of inherited SYS-1. Photoconversion during mitosis reveals that SYS-1 clearance at the centrosome preferentially degrades older SYS-1 and that newly localized centrosomal SYS-1 depends on dynein trafficking. Photoconversion of DENDRA2::SYS-1 in the EMS cell during Wnt-driven ACD shows daughter cell inheritance of mother cell SYS-1. Additionally, disrupting centrosomal SYS-1 localization in mother cells increased inherited SYS-1 and, surprisingly, loss of centrosomal SYS-1 also resulted in increased levels of de novo SYS-1 in both EMS daughter cells. Lastly, we show that negative regulation of SYS-1 in daughter cells via the destruction complex member APR-1/APC is key to limit both the de novo and the inherited SYS-1 pools in both the E and the MS cells. We conclude that regulation of both inherited and newly translated SYS-1 via centrosomal processing in the mother cell and daughter cell regulation via Wnt signaling are critical to maintain sister SYS-1 asymmetry during ACD.

DOI: https://doi.org/10.1091/mbc.E24-10-0441
ACS Pharmacol Transl Sci. 2025 Jan 10. 8(1): 21-35

Activation of the 26S Proteasome to Reduce Proteotoxic Stress and Improve the Efficacy of PROTACs.

Jindrich Sedlacek.

The 26S proteasome degrades the majority of cellular proteins and affects all aspects of cellular life. Therefore, the 26S proteasome abundance, proper assembly, and activity in different life contexts need to be precisely controlled. Impaired proteasome activity is considered a causative factor in several serious disorders. Recent advances in proteasome biology have revealed that the proteasome can be activated by different factors or small molecules. Thus, activated ubiquitin-dependent proteasome degradation has effects such as extending the lifespan in different models, preventing the accumulation of protein aggregates, and reducing their negative impact on cells. Increased 26S proteasome-mediated degradation reduces proteotoxic stress and can potentially improve the efficacy of engineered degraders, such as PROTACs, particularly in situations characterized by proteasome malfunction. Here, emerging ideas and recent insights into the pharmacological activation of the proteasome at the transcriptional and posttranslational levels are summarized.

DOI: https://doi.org/10.1021/acsptsci.4c00408
Transl Oncol. 2025 Jan 14. pii: S1936-5233(25)00002-6. [Epub ahead of print]52 102271

Combined anti-leukemic effect of gilteritinib and GSK-J4 in FLT3-ITD+ acute myeloid leukemia.

Qi Zhou, Yongyu Guan, Pingping Zhao, Huiyuan Chu, Yaming Xi.

  Gilteritinib treats acute myeloid leukemia (AML) with the FMS-like receptor tyrosine kinase-3 (FLT3) internal tandem duplication (ITD) mutation. Dysregulation of histone modification affects the genesis and progression of AML. Strategies targeting key histone regulators have not been applied to the treatment of AML. Lysine demethylase 6B (KDM6B) is dysregulated in a variety of cancers and regulates the expression of oncogenes, which has potential in anticancer therapy. We explored whether GSK-J4 (an inhibitor of the demethylase KDM6B) has an anti-leukemic effect in the gilteritinib treatment of FLT3-ITD+ AML and the effect of gilteritinib combined with GSK-J4 in leukemia. In our study, we evaluated the anti-leukemic effect of GSK-J4 in gilteritinib therapy through in vitro and in vivo experiments. The results revealed that the combined treatment of gilteritinib and GSK-J4 has greater anti-proliferation and pro-apoptosis effects than gilteritinib alone. Gilteritinib and GSK-J4 performed synergistically to arrest the cell cycle. Gilteritinib mainly induces cell cycle phase arrest at the S or G0/G1, and GSK-J4 inhibits the cell cycle progression in the S phase and reduces cell viability by reducing the expression of key regulatory factors from the G1 phase to the S phase. At the same time, GSK-J4 enhances the expression of apoptosis-related proteins (Bax and cleavage caspase-9). In addition, gilteritinib or GSK-J4 monotherapy increases reactive oxygen species (ROS) production, and the combination has a synergistic effect, accelerating leukemic cell death. Our study provides proof that the combined therapy of gilteritinib and GSK-J4 has a synergistic antileukemic effect on FLT3-ITD+ AML.

Keywords:  Acute myeloid leukemia; Combination therapy; FLT3-ITD; GSK-J4; Gilteritinib

DOI:  https://doi.org/10.1016/j.tranon.2025.102271