Long-term persistence of chimeric antigen receptor (CAR)-T cells is essential for durable therapeutic efficacy but the mechanisms underlying CAR-T cell dysfunction remain unclear. On the basis of integrated analyses of clinical samples from participants with multiple myeloma and acute lymphoblastic leukemia treated with CAR-T cells, we show that rapid expansion of CAR-T cells after infusion is followed by a 'diminution' phase characterized by ferroptosis-associated features and elevated serum iron levels. In preclinical cancer models in female mice and ex vivo culture systems, excess intracellular iron impaired CAR-T cell function. Mechanistically, iron promoted ferroptosis by increasing mitochondrial reactive oxygen species and lipid peroxidation, in part through acyl-CoA synthetase long-chain family member 4 (ACSL4)-associated lipid remodeling. Targeting ferroptosis, particularly through genetic ablation of ACSL4 in CAR-T cells, substantially enhanced antitumor efficacy. Together, these findings identify iron-driven ferroptosis as a determinant of CAR-T cell dysfunction and a targetable barrier to durable CAR-T efficacy.

Human embryonic stem cells (hESCs) serve as an ideal cell source for generating hematopoietic stem cells (HSCs). In embryonic hematopoiesis, hemogenic endothelium has been identified as a source of HSCs, yet the regulatory mechanisms remain elusive. Here, through dynamic gene expression profiling analysis and verification, we find that ELTD1 expression parallels genes related to the specification of hemogenic endothelium progenitors (HEPs) from hESCs and is highly expressed in the HEPs. We then investigate the impact of ELTD1 on the hematopoietic differentiation of hESCs via gain- and loss-of-function experiments. Knockdown or deletion of ELTD1 mediates hESC hematopoiesis by specifically facilitating the generation of HEPs, thus promoting endothelial-to-hematopoietic transition to generate more hematopoietic cells. Besides, the overexpression of ELTD1 serves to further solidify this conclusion. Mechanistically, we demonstrate that ELTD1 exerts its function through the Wnt signaling pathway by bioinformatic analyses and functional studies. In addition, our results demonstrate a protein-protein interaction between ELTD1 and HPIP and further reveal that HPIP modulates the Wnt signaling pathway through LEF1. Collectively, these findings indicate that the ELTD1-HPIP-LEF1-Wnt regulatory axis acts as a novel mechanism regulating HEP generation during early hematopoietic differentiation of hESCs, providing new insights into the molecular mechanisms underlying human hematopoiesis.

Clinical evidence supports the notion that T cell exhaustion and terminal differentiation pose challenges to the persistence and effectiveness of chimeric antigen receptor-T (CAR-T) cells. MEK1/2 inhibitors (MEKIs), widely used in cancer treatment due to their ability to inhibit aberrant MAPK signaling, have shown potential synergistic effects when combined with immunotherapy. However, the impact and mechanisms of MEKIs on CAR-T cells remain uncertain and controversial. To address this, we conducted a comprehensive investigation to determine whether MEKIs enhance or impair the efficacy of CAR-T cells. Our findings revealed that MEKIs attenuated CAR-T cell exhaustion and terminal differentiation induced by tonic signaling and antigen stimulation, thereby improving CAR-T cell efficacy against hematological and solid tumors. Remarkably, these effects were independent of the specific scFvs and costimulatory domains utilized in CARs. Mechanistically, analysis of bulk and single-cell transcriptional profiles demonstrates that the effect of MEK inhibition was related to diminish anabolic metabolism and downregulation of c-Fos and JunB. Additionally, the overexpression of c-Fos or JunB in CAR-T cells counteracted the effects of MEK inhibition. Furthermore, our Cut-and-Tag assay revealed that MEK inhibition downregulated the JunB-driven gene profiles associated with exhaustion, differentiation, anergy, glycolysis, and apoptosis. In summary, our research unveil the critical role of the MAPK-c-Fos-JunB axis in driving CAR-T cell exhaustion and terminal differentiation. These mechanistic insights significantly broaden the potential application of MEKIs to enhance the effectiveness of CAR-T therapy.

Aging of hematopoietic stem cells (HSCs) is accompanied by impaired self-renewal ability, myeloid skewing, immunodeficiencies and increased susceptibility to malignancies. Although previous studies highlighted the pivotal roles of individual metabolites in hematopoiesis, comprehensive and high-resolution metabolomic profiles of different hematopoietic cells across ages are still lacking. In this study, we created a metabolome atlas of different blood cells across ages in mice. We reveal here that purine, pyrimidine and retinol metabolism are enriched in young hematopoietic stem and progenitor cells (HSPCs), whereas glutamate and sphingolipid metabolism are concentrated in aged HSPCs. Through metabolic screening, we identified uridine as a potential regulator to rejuvenate aged HSPCs. Mechanistically, uridine treatment upregulates the FoxO signaling pathway and enhances self-renewal while suppressing inflammation in aged HSCs. Finally, we constructed an open-source platform for public easy access and metabolomic analysis in blood cells. Collectively, we provide a resource for metabolic studies in hematopoiesis that can contribute to future anti-aging metabolite screening.

Figure S3. Metastatic tumor specific genes

Figure S7. Similarity and variation of lncRNA profiles in single cells.

Figure S2. Comparison of the expression of lncRNAs and PCGs in single cells and bulk tissues.

Figure S4. 173 differentially expressed lncRNAs profiling of all single cells.

Hematopoietic stem cell (HSC) aging is accompanied by hematopoietic reconstitution dysfunction, including loss of regenerative and engraftment ability, myeloid differentiation bias, and elevated risks of hematopoietic malignancies. Gut microbiota, a key regulator of host health and immunity, has recently been reported to affect hematopoiesis. However, there is currently limited empirical evidence explaining the direct impact of gut microbiome on aging hematopoiesis. In this study, we performed fecal microbiota transplantation (FMT) from young mice to aged mice and observed a significant increment in lymphoid differentiation and decrease in myeloid differentiation in aged recipient mice. Furthermore, FMT from young mice rejuvenated aged HSCs with enhanced short-term and long-term hematopoietic repopulation capacity. Mechanistically, single-cell RNA sequencing deciphered that FMT from young mice mitigated inflammatory signals, upregulated the FoxO signaling pathway, and promoted lymphoid differentiation of HSCs during aging. Finally, integrated microbiome and metabolome analyses uncovered that FMT reshaped gut microbiota composition and metabolite landscape, and Lachnospiraceae and tryptophan-associated metabolites promoted the recovery of hematopoiesis and rejuvenated aged HSCs. Together, our study highlights the paramount importance of the gut microbiota in HSC aging and provides insights into therapeutic strategies for aging-related hematologic disorders.

Table S1. Information of RNA-seq samples. Table S2. Metastatic vs Primary differentially expressed lncRNAs and PCGs. Table S3. Correlation between CMAL-clusters and clinicopathological features. Table S4. CMALs significantly associated with TNF signaling pathway. Table S5. The qRT-PCR primer sequences of four CMALs and two PCGs.

Figure S5. The proportion of TCGA CMAL-clusters associated with TCGA clinical features and mRNA-, miRNA- clusters.

Figure S1. Expression of lncRNAs and PCGs in single cells and bulk tissues

Objectives:Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy with widely variable prognosis. For this reason, a more tailored-stratified approach for prognosis is urgently needed to improve the treatment success rates of AML patients.Methods:In the investigation of metabolic pattern in AML patients, we developed a metabolism-related prognostic model, which was consisted of metabolism-related gene pairs (MRGPs) identified by pairwise comparison. Furthermore, we analyzed the predictive ability and clinical significance of the prognostic model.Results:Given the significant differences in metabolic pathways between AML patients and healthy donors, we proposed a metabolism-related prognostic signature index (MRPSI) consisting of three MRGPs, which were remarkedly related with the overall survival of AML patients in the training set. The association of MRPSI with prognosis was also validated in two other independent cohorts, suggesting that high MRPSI score can identify patients with poor prognosis. The MRPSI and age were confirmed to be independent prognostic factors via multivariate Cox regression analysis. Furthermore, we combined MRPSI with age and constructed a composite metabolism-clinical prognostic model index (MCPMI), which demonstrated better prognostic accuracy in all cohorts. Stratification analysis and multivariate Cox regression analysis revealed that the MCPMI was an independent prognostic factor. By estimating the sensitivity of anti-cancer drugs in different AML patients, we selected five drugs that were more sensitive to patients in MCPMI-high group than those in MCPMI-low group.Conclusion:Our study provided an individualized metabolism-related prognostic model that identified high-risk patients and revealed new potential therapeutic drugs for AML patients with poor prognosis.

Introduction: The effects of MEK inhibitors (MEKIs) in CAR-T cells are poorly understood and remain controversial. Some groups showed that MEKIs could impair CAR-T cells' function in vitro. However, another group reported that MEKIs could combine with GD2 CAR-T cells to provide additional efficacy. Yet, the mechanism for this combined effect is unclear. Considering that the CAR signaling pathway is similar to TCR, both involve the activation of MAPK signaling. We hypothesized that MEKIs might mitigate CAR-T cells' exhaustion and terminal differentiation by lessening redundant CAR signaling. The study aims to systematically evaluate the role and mechanism of MEK inhibitors on CAR-T cells. Methods and Results: To explore whether MEKIs could mitigate the unbeneficial impact of antigen-independent CAR tonic signaling, we added 3 FDA-approved MEKIs: trametinib, cobimetinib, and binimetinib, respectively, to the culture medium of CD19.28z CAR-T cells for 9 days at the concentration approaching their clinically tolerable peak blood concentration. We found that all the 3 MEKIs could reduce CAR-T cells' terminal differentiation and restrain the expression of exhaustion and activation markers. Among them, trametinib was the most potent because it could achieve the comparable effect of cobimetinib and binimetinib at the lowest concentration. Thus, we chose trametinib for further research. We confirmed that compared to concentrations of 7.5nM and 30nM, 15nM was the optimal concentration of trametinib with mild inhibition on the proliferation of CAR-T cells and a potent effect on CAR-T cells' phenotype. Pre-treatment with trametinib didn't affect the in-vitro cytotoxicity of CD19.28z CAR-T cells. The above effects of trametinib were more significant as treatment time and the dose increased. Similar results could be obtained in analogous experiments with CD19.4-1BBz and GD2.28z CAR-T cells. Intriguingly, GD2.28z CAR-T cells cultured with trametinib had better proliferation and killing capacity because they were more exhausted than CD19.28z CAR-T cells. Using the Nalm-6-bearing leukemia xenograft model, we found that compared to DMSO pre-treated CD19.28z CAR-T cells, trametinib pre-treated CD19.28z CAR-T cells exerted more potent anti-leukemia activity, showed a less exhausted and differentiated state, proliferated better, and further extended mice survival (Fig.1). Similar results could be obtained in analogous in-vivo experiments with CD19.4-1BBz CAR-T cells. To evaluate whether trametinib could protect CAR-T cells from the exhaustion and terminal differentiation triggered by antigen stimulation, we cocultured CD19.28z CAR-T cells with Nalm-6 cells in a medium with or without trametinib. We demonstrated under antigen stimulation, trametinib could effectively inhibit CAR-T cells' activation, exhaustion, apoptosis, terminal differentiation, and phosphorylation of ERK and consequently promote the proliferation of total and CD8 CAR-T cells. In addition, after repetitive antigen stimulation, trametinib could rescue CAR-T cells' functional exhaustion and improve CAR-T cells' in-vitro cytotoxicity. Mechanistically, Single-cell and bulk RNA-Seq revealed that the effect of MEK inhibition was associated with the downregulation of AP-1 and exhaustion-associated transcription factors (TFs) and the upregulation of memory-associated TFs (Fig.2). GSEA revealed the upregulation of naive/memory-associated genes and downregulation of genes involved in T cell activation/effector/exhaustion, AP-1 pathway and apoptosis in the trametinib-treated group. Additionally, single-cell transcriptional profiling demonstrated enrichment of memory and Ki67+cycling CAR-T clusters in the trametinib-treated group. Among the AP-1 TFs downregulated by trametinib, c-Fos and JunB are the direct downstream targets of the canonical MAPK signaling pathway, and both of them were reported to implicate T cell exhaustion. Thus we speculated both downregulation of c-Fos and JunB may contribute to the role of MEKIs. Consistent with our hypothesis, overexpression of c-Fos or JunB in CAR-T cells could partially, if not all, abrogated the effects of MEK inhibition. Significance: Our research provides a strategy to optimize CAR-T antitumor efficacy by MEK inhibition and shed light on the role of c-Fos and JunB in driving CAR-T cell exhaustion and terminal differentiation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Myeloid-derived suppressor cells (MDSCs) represent a population of heterogeneous myeloid cells, which are characterized by their remarkable ability to suppress T cells and natural killer cells. MDSCs have been proven to play a positive role in protecting acute graft-versus-host disease (aGVHD). Here, we aimed to describe the mechanism behind how mTOR signaling regulates MDSCs’ generation and explore its prophylactic and therapeutic potential in aGVHD. Reducing mTOR expression retains myeloid cells with immature characteristics and promotes polymorphonuclear MDSC (PMN-MDSC) immunosuppressive function through STAT3-C/EBPβ pathway. Prophylactic transfusion of mTORKO PMN-MDSCs could alleviate aGVHD while maintaining the graft-versus-leukemia (GVL) effect, which could downregulate the Th1/Th2 ratio, decrease serum proinflammatory cytokines, and increase the proportion of regulatory T cells (Tregs) in aGVHD models at the early stage after transplantation. Moreover, transfusion therapy could promote the reconstruction and function of donor-derived PMN-MDSCs. Not only the percentage and the absolute number of donor-derived PMN-MDSCs significantly increased but also the immunosuppressive ability was much more robust compared to other groups. Altogether, these findings indicated that mTOR is an intrinsic regulator for PMN-MDSCs’ differentiation and immunosuppressive function. Together, mTORKO PMN-MDSC transfusion can play a protective role in alleviating cytokine storm at the initial stage and promoting the quantitative and functional recoveries of donor-derived PMN-MDSCs in aGVHD.

Hematopoietic stem cell (HSC) aging, which is accompanied by loss of self-renewal capacity, myeloid-biased differentiation and increased risks of hematopoietic malignancies, is an important focus in stem cell research. However, the mechanisms underlying HSC aging have not been fully elucidated. In the present study, we integrated 3 independent single-cell transcriptome datasets of HSCs together and identified Stat3 and Ifngr1 as two markers of apoptosis-biased and inflammatory aged HSCs. Besides, common differentially expressed genes (DEGs) between young and aged HSCs were identified and further validated by quantitative RT-PCR. Functional enrichment analysis revealed that these DEGs were predominantly involved in the cell cycle and the tumor necrosis factor (TNF) signaling pathway. We further found that the Skp2-induced signaling pathway (Skp2→Cip1→CycA/CDK2→DP-1) contributed to a rapid transition through G1 phase in aged HSCs. In addition, analysis of the extrinsic alterations on HSC aging revealed the increased expression levels of inflammatory genes in bone marrow microenvironment. Colony formation unit assays showed that inflammatory cytokines promoted cellular senescence and that blockade of inflammatory pathway markedly rejuvenated aged HSC functions and increased B cell output. Collectively, our study elucidated the biological characteristics of HSC aging, and the genes and pathways we identified could be potential biomarkers and targets for the identification and rejuvenation of aged HSCs.

Human ELTD1 (Epidermal growth factor, latrophilin and seven-transmembrane domain-containing 1), an orphan G-protein-coupled receptor (GPCR) belonging to the adhesion GPCR family, has been reported as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target. However, little is known about the function of ELTD1, especially its undiscovered ligands. In this experiment, an ELTD1 homozygous knockout human embryonic stem cell line, FAHZUe001-A, was generated by the iCRISPR/Cas9 system to achieve a deeper understanding of ELTD1. The FAHZUe001-A was confirmed with normal karyotype, typical undifferentiated morphology, pluripotency and trilineage differentiation potential in vitro.

Fat mass and obesity-associated protein (FTO) is the first protein found to have the activity of N6-methyladenosine (m6A) demethylation. It has been reported that FTO was involved in different physiological and pathological processes, including stem cell differentiation, sex determination, tumorigenesis, and progression. To further understand the exact role of FTO in these processes, we generated a FTO knockout human embryonic stem cell (hESC) line by CRISPR/Cas9 mediated gene editing method. This cell line maintained normal karyotype, pluripotency, and trilineage differentiation potential, which are considered as a model for function studies of the FTO protein in hESC self-renewal and differentiation.

Widely known for self-renewal and multilineage differentiation, stem cells can be differentiated into all specialized tissues and cells in the body. In the past few years, a number of researchers have focused on deriving hematopoietic stem cells (HSCs) from pluripotent stem cells (PSCs) as alternative sources for clinic. Existing findings demonstrated that it is feasible to obtain HSCs and certain mature blood lineages from PSCs, except for several issues to be addressed. This short review outlines the technologies used for hematopoietic differentiation in recent years. In addition, the therapeutic value of PSCs as a potential source of various blood cells is also discussed as well as its challenges and directions in future clinical applications.