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Breast cancer stem cell (CSC) expansion results in tumor progression and chemoresistance; however,the modulation of CSC pluripotency remains unexplored. Transmembrane protein 120B (TMEM120B) is a newly discovered protein expressed in human tissues,especially in malignant tissues; however,its role in CSC expansion has not been studied. This study aimed to determine the role of TMEM120B in transcriptional coactivator with PDZ-binding motif (TAZ)-mediated CSC expansion and chemotherapy resistance. Both bioinformatics analysis and immunohistochemistry assays were performed to examine expression patterns of TMEM120B in lung,breast,gastric,colon,and ovarian cancers. Clinicopathological factors and overall survival were also evaluated. Next,colony formation assay,MTT assay,EdU assay,transwell assay,wound healing assay,flow cytometric analysis,sphere formation assay,western blotting analysis,mouse xenograft model analysis,RNA-sequencing assay,immunofluorescence assay,and reverse transcriptase-polymerase chain reaction were performed to investigate the effect of TMEM120B interaction on proliferation,invasion,stemness,chemotherapy sensitivity,and integrin/FAK/TAZ/mTOR activation. Further,liquid chromatography–tandem mass spectrometry analysis,GST pull-down assay,and immunoprecipitation assays were performed to evaluate the interactions between TMEM120B,myosin heavy chain 9 (MYH9),and CUL9. TMEM120B expression was elevated in lung,breast,gastric,colon,and ovarian cancers. TMEM120B expression positively correlated with advanced TNM stage,lymph node metastasis,and poor prognosis. Overexpression of TMEM120B promoted breast cancer cell proliferation,invasion,and stemness by activating TAZ-mTOR signaling. TMEM120B directly bound to the coil-coil domain of MYH9,which accelerated the assembly of focal adhesions (FAs) and facilitated the translocation of TAZ. Furthermore,TMEM120B stabilized MYH9 by preventing its degradation by CUL9 in a ubiquitin-dependent manner. Overexpression of TMEM120B enhanced resistance to docetaxel and doxorubicin. Conversely,overexpression of TMEM120B-∆CCD delayed the formation of FAs,suppressed TAZ-mTOR signaling,and abrogated chemotherapy resistance. TMEM120B expression was elevated in breast cancer patients with poor treatment outcomes (Miller/Payne grades 1–2) than in those with better outcomes (Miller/Payne grades 3–5). Our study reveals that TMEM120B bound to and stabilized MYH9 by preventing its degradation. This interaction activated the β1-integrin/FAK-TAZ-mTOR signaling axis,maintaining stemness and accelerating chemotherapy resistance. The online version contains supplementary material available at 10.1186/s13058-024-01802-z. View Publication

Chimeric antigen receptor (CAR) T cells have shown remarkable response rates in hematological malignancies. In contrast,CAR T cell treatment of solid tumors is associated with several challenges,in particular the expression of most tumor-associated antigens at lower levels in vital organs,resulting in on-target/off-tumor toxicities. Thus,innovative approaches to improve the tumor specificity of CAR T cells are urgently needed. Based on the observation that many human solid tumors activate epidermal growth factor receptor (EGFR) on their surface through secretion of EGFR ligands,we developed an engineering strategy for CAR-binding domains specifically directed against the ligand-activated conformation of EGFR. We show,in several experimental systems,that the generated binding domains indeed enable CAR T cells to distinguish between active and inactive EGFR. We anticipate that this engineering concept will be an important step forward to improve the tumor specificity of CAR T cells directed against EGFR-positive solid cancers. View Publication

In the adult bone marrow (BM),endothelial cells (ECs) are an integral component of the hematopoietic stem cell (HSC)-supportive niche,which modulates HSC activity by producing secreted and membrane-bound paracrine signals. Within the BM,distinct vascular arteriole,transitional,and sinusoidal EC subtypes display unique paracrine expression profiles and create anatomically-discrete microenvironments. However,the relative contributions of vascular endothelial subtypes in supporting hematopoiesis is unclear. Moreover,constitutive expression and off-target activity of currently available endothelial-specific and endothelial-subtype-specific murine cre lines potentially confound data analysis and interpretation. To address this,we describe two tamoxifen-inducible cre -expressing lines,Vegfr3-creER T2 and Cx40-creER T2,that efficiently label sinusoidal/transitional and arteriole endothelium respectively in adult marrow,without off-target activity in hematopoietic or perivascular cells. Utilizing an established mouse model in which cre -dependent recombination constitutively-activates MAPK signaling within adult endothelium,we identify arteriole ECs as the driver of MAPK-mediated hematopoietic dysfunction. These results define complementary tamoxifen-inducible creER T2 -expressing mouse lines that label functionally-discrete and non-overlapping sinusoidal/transitional and arteriole EC populations in the adult BM,providing a robust toolset to investigate the differential contributions of vascular subtypes in maintaining hematopoietic homeostasis. The online version contains supplementary material available at 10.1007/s12015-024-10703-9. View Publication

The human WDR33 gene encodes three major isoforms. The canonical isoform WDR33v1 (V1) is a well-characterized nuclear mRNA polyadenylation factor,while the other two,WDR33v2 (V2) and WDR33v3 (V3),have not been studied. Here,we report that V2 and V3 are generated by alternative polyadenylation,and neither protein contains all seven WD (tryptophan-aspartic acid) repeats that characterize V1. Surprisingly,V2 and V3 are not polyadenylation factors but localize to the endoplasmic reticulum and interact with stimulator of interferon genes (STING),the immune factor that induces the cellular response to cytosolic double-stranded DNA. V2 suppresses interferon-β induction by preventing STING disulfide oligomerization but promotes autophagy,likely by recruiting WIPI2 isoforms. V3,on the other hand,functions to ncrease STING protein levels. Our study has not only provided mechanistic insights into STING regulation but also revealed that protein isoforms can be functionally completely unrelated,indicating that alternative mRNA processing is a more powerful mechanism than previously appreciated. View Publication

Wiskott-Aldrich syndrome (WAS) is a severe X-linked primary immunodeficiency resulting from a diversity of mutations distributed across all 12 exons of the WAS gene. WAS encodes a hematopoietic-specific and developmentally regulated cytoplasmic protein (WASp). The objective of this study was to develop a gene correction strategy potentially applicable to most WAS patients by employing nuclease-mediated,site-specific integration of a corrective WAS gene sequence into the endogenous WAS chromosomal locus. In this study,we demonstrate the ability to target the integration of WAS 2-12 -containing constructs into intron 1 of the endogenous WAS gene of primary CD34 + hematopoietic stem and progenitor cells (HSPCs),as well as WASp-deficient B cell lines and WASp-deficient primary T cells. This intron 1 targeted integration (TI) approach proved to be quite efficient and restored WASp expression in treated cells. Furthermore,TI restored WASp-dependent function to WAS patient T cells. Edited CD34 + HSPCs exhibited the capacity for multipotent differentiation to various hematopoietic lineages in vitro and in transplanted immunodeficient mice. This methodology offers a potential editing approach for treatment of WAS using patient’s CD34 + cells. View Publication

Current anticancer therapies cannot eliminate all cancer cells,which hijack normal arginine methylation as a means to promote their maintenance via unknown mechanisms. Here we show that targeting protein arginine N -methyltransferase 9 (PRMT9),whose activities are elevated in blasts and leukemia stem cells (LSCs) from patients with acute myeloid leukemia (AML),eliminates disease via cancer-intrinsic mechanisms and cancer-extrinsic type I interferon (IFN)-associated immunity. PRMT9 ablation in AML cells decreased the arginine methylation of regulators of RNA translation and the DNA damage response,suppressing cell survival. Notably,PRMT9 inhibition promoted DNA damage and activated cyclic GMP-AMP synthase,which underlies the type I IFN response. Genetically activating cyclic GMP-AMP synthase in AML cells blocked leukemogenesis. We also report synergy of a PRMT9 inhibitor with anti-programmed cell death protein 1 in eradicating AML. Overall,we conclude that PRMT9 functions in survival and immune evasion of both LSCs and non-LSCs; targeting PRMT9 may represent a potential anticancer strategy. Subject terms: Cancer,Tumour immunology View Publication

Hematopoietic stem cells (HSCs) produce all essential cellular components of the blood. Stromal cell lines supporting HSCs follow a vascular smooth muscle cell (vSMC) differentiation pathway,suggesting that some hematopoiesis-supporting cells originate from vSMC precursors. These pericyte-like precursors were recently identified in the aorta-gonad-mesonephros (AGM) region; however,their role in the hematopoietic development in vivo remains unknown. Here,we identify a subpopulation of NG2 + Runx1 + perivascular cells that display a sclerotome-derived vSMC transcriptomic profile. We show that deleting Runx1 in NG2 + cells impairs the hematopoietic development in vivo and causes transcriptional changes in pericytes/vSMCs,endothelial cells and hematopoietic cells in the murine AGM. Importantly,this deletion leads also to a significant reduction of HSC reconstitution potential in the bone marrow in vivo. This defect is developmental,as NG2 + Runx1 + cells were not detected in the adult bone marrow,demonstrating the existence of a specialised pericyte population in the HSC-generating niche,unique to the embryo. Subject terms: Cell biology,Haematopoiesis,Cardiovascular biology View Publication

Understanding why some triple-negative breast cancer (TNBC) patients respond poorly to existing therapies while others respond well remains a challenge. This study aims to understand the potential underlying mechanisms distinguishing early-stage TNBC tumors that respond to clinical intervention from non-responders,as well as to identify clinically viable therapeutic strategies,specifically for TNBC patients who may not benefit from existing therapies. We conducted retrospective bioinformatics analysis of historical gene expression datasets to identify a group of genes whose expression levels in early-stage tumors predict poor clinical outcomes in TNBC. In vitro small-molecule screening,genetic manipulation,and drug treatment in syngeneic mouse models of TNBC were utilized to investigate potential therapeutic strategies and elucidate mechanisms of drug action. Our bioinformatics analysis reveals a robust association between increased expression of immunosuppressive cytokine S100A8/A9 in early-stage tumors and subsequent disease progression in TNBC. A targeted small-molecule screen identifies PIM kinase inhibitors as capable of decreasing S100A8/A9 expression in multiple cell types,including TNBC and immunosuppressive myeloid cells. Combining PIM inhibition and immune checkpoint blockade induces significant antitumor responses,especially in otherwise resistant S100A8/A9-high PD-1/PD-L1-positive tumors. Notably,serum S100A8/A9 levels mirror those of tumor S100A8/A9 in a syngeneic mouse model of TNBC. Our data propose S100A8/A9 as a potential predictive and pharmacodynamic biomarker in clinical trials evaluating combination therapy targeting PIM and immune checkpoints in TNBC. This work encourages the development of S100A8/A9-based liquid biopsy tests for treatment guidance. Subject terms: Breast cancer,Breast cancer,Prognostic markers View Publication

Ferroptosis is a new form of nonapoptotic and iron-dependent type of cell death. Glutathione peroxidase-4 (GPX4) plays an essential role in anti-ferroptosis by reducing lipid peroxidation. Although acute myeloid leukemia (AML) cells,especially relapsed and refractory (R/R)-AML,present high GPX4 levels and enzyme activities,pharmacological inhibition of GPX4 alone has limited application in AML. Thus,whether inhibition of GPX4 combined with other therapeutic reagents has effective application in AML is largely unknown. Lipid reactive oxygen species (ROS),malondialdehyde (MDA),and glutathione (GSH) assays were used to assess ferroptosis in AML cells treated with the hypomethylating agent (HMA) decitabine (DAC),ferroptosis-inducer (FIN) RAS-selective lethal 3 (RSL3),or their combination. Combination index (CI) analysis was used to assess the synergistic activity of DAC + RSL3 against AML cells. Finally,we evaluated the synergistic activity of DAC + RSL3 in murine AML and a human R/R-AML-xenografted NSG model in vivo. We first assessed GPX4 expression and found that GPX4 levels were higher in AML cells,especially those with MLL rearrangements,than in NCs. Knockdown of GPX4 by shRNA and indirect inhibition of GPX4 enzyme activity by RSL3 robustly induced ferroptosis in AML cells. To reduce the dose of RSL3 and avoid side effects,low doses of DAC (0.5 µM) and RSL3 (0.05 µM) synergistically facilitate ferroptosis by inhibiting the AMP-activated protein kinase (AMPK)-SLC7A11-GPX4 axis. Knockdown of AMPK by shRNA enhanced ferroptosis,and overexpression of SLC7A11 and GPX4 rescued DAC + RSL3-induced anti-leukemogenesis. Mechanistically,DAC increased the expression of MAGEA6 by reducing MAGEA6 promoter hypermethylation. Overexpression of MAGEA6 induced the degradation of AMPK,suggesting that DAC inhibits the AMPK-SLC7A11-GPX4 axis by increasing MAGEA6 expression. In addition,DAC + RSL3 synergistically reduced leukemic burden and extended overall survival compared with either DAC or RSL3 treatment in the MLL-AF9-transformed murine model. Finally,DAC + RSL3 synergistically reduced viability in untreated and R/R-AML cells and extended overall survival in two R/R-AML-xenografted NSG mouse models. Our study first identify vulnerability to ferroptosis by regulating MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway. Combined treatment with HMAs and FINs provides a potential therapeutic choice for AML patients,especially for R/R-AML. The online version contains supplementary material available at 10.1186/s40164-024-00489-4. View Publication

Chronic myelogenous leukemia (CML) is a clonal hematologic malignancy of the myeloid lineage caused by the oncogenic BCR/ABL fusion protein that promotes CML cell proliferation and protects them against drug-induced apoptosis. In this study,we determine LATS1 and LATS2 expression in CML cells derived from patients who are resistant to imatinib (IM) treatment. Significant upregulation of LATS1 and LATS2 was found in these CML patients compared to healthy donors. To further explore whether the expression of LATS1/2 contributes to the IM-resistant phenotype,IM-resistant CML cell lines generated by culturing CML-derived erythroblastic K562 cells in increasing concentrations of IM were used as in vitro models. Up-regulation of LATS1 and LATS2 was observed in IM-resistant K562 cells. Reduction of LATS using either Lats-IN-1 (TRULI),a specific LATS inhibitor,or shRNA targeting LATS1/2 significantly reduced clonogenicity,increased apoptosis and induced differentiation of K562 cells to late-stage erythroid cells. Furthermore,depletion of LATS1 and LATS2 also increased the sensitivity of K562 cells to IM. Taken together,our results suggest that LATS could be one of the key factors contributing to the rapid proliferation,reduced apoptosis,and IM resistance of CML cells. Targeting LATS could be a promising treatment to enhance the therapeutic effect of a conventional BCR/ABL tyrosine kinase inhibitor such as IM. View Publication

The development of vascular networks in microfluidic chips is crucial for the long-term culture of three-dimensional cell aggregates such as spheroids,organoids,tumoroids,or tissue explants. Despite rapid advancement in microvascular network systems and organoid technologies,vascularizing organoids-on-chips remains a challenge in tissue engineering. Most existing microfluidic devices poorly reflect the complexity of in vivo flows and require complex technical set-ups. Considering these constraints,we develop a platform to establish and monitor the formation of endothelial networks around mesenchymal and pancreatic islet spheroids,as well as blood vessel organoids generated from pluripotent stem cells,cultured for up to 30 days on-chip. We show that these networks establish functional connections with the endothelium-rich spheroids and vascular organoids,as they successfully provide intravascular perfusion to these structures. We find that organoid growth,maturation,and function are enhanced when cultured on-chip using our vascularization method. This microphysiological system represents a viable organ-on-chip model to vascularize diverse biological 3D tissues and sets the stage to establish organoid perfusions using advanced microfluidics. Subject terms: Stem-cell biotechnology,Tissue engineering,Biomedical engineering,Induced pluripotent stem cells,Microfluidics View Publication

RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer’s disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology,we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein,we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM,however,these compounds lack selectivity and this limits their utility as chemical tools. View Publication