all metabolite intensities(mouse model)

all metabolite intensities(patients)

Supplementary methods, tables,figures and figure legends

Increasing numbers of clinical cohorts have detected CCND2 mutations in acute myeloid leukemia (AML), especially in the subtype of AML with t(8;21) translocation. As known, this AML subtype is characterized by the formation of AML1-ETO fusion gene. However, AML1-ETO fusion gene alone is not sufficient to drive leukemia development, additional mutations are required for leukemogenesis. In this study, we aim to investigate whether mutated CCND2 can cooperate with AML1-ETO fusion gene to drive leukemia initiation and progression. In our previous study, the conditional AML1-ETO knock-in mouse model (AML1/ETO mouse), which represented a pre-leukemia stage as myeloproliferative neoplasm phenotype, was established. To confirm whether the AML1-ETO and CCND2 mutation can cooperate to drive leukemia, the mice transduction and transplantation model harboring both AML1-ETO and CCND2 gene (both wildtype and mutant) were established. Upon the assessment of the phenotype, biological features and survival of the mice, only the mice overexpressing the AML1-ETO and CCND2 simultaneously were eventually progressed to leukemia. Besides, compared to mice overexpressing AML-ETO gene alone, mTOR and cell cycle-related pathways were significantly enriched in mice harboring both AML1-ETO and CCND2. And the selective mTOR inhibitor, Everolimus, can reduce the leukemia burden and prolong the survival of this group of mice. In conclusion, it was confirmed that introduction of the CCND2 gene into the AML/ETO pre-leukemia mice could trigger the development of leukemia. It was also confirmed that CCND2 overexpression resulted in the upregulation of the mTOR pathway and inhibiting the pathway might be a therapeutic strategy for this subtype of leukemia.

The t(8;21)(q22;q22) translocation is one of the most recurrent cytogenetic aberrations in acute myeloid leukaemia (AML). While most patients achieve complete remission, approximately 40% of them still relapse. Early identification and elimination of leukaemia clones with relapse potential could improve prognosis for t(8;21) AML patients. Here, through single-cell RNA sequencing, we characterized the intra-tumoral heterogeneity of t(8;21) AML and identified haematopoietic stem cell (HSC)-like subset as the most quiescent and primitive subgroup among all leukaemia cell populations. Further investigations revealed IKZF2 as the master regulator for HSC-like subset. Notably, IKZF2 was upregulated in t(8;21) AML compared with other AML subtypes and was specifically targeted by AML1-ETO. Using primary samples and mouse models, we verified the high enrichment of IKZF2 in primitive and quiescent leukaemic cells. Moreover, IKZF2 knockout hindered the accumulation of aberrant stem cells driven by AML1-ETO and promoted cellular differentiation both in vitro and in vivo. These facilitate a better understanding of the leukaemia cell heterogeneity in t(8;21) AML and unveil IKZF2 as a potential target for improving current treatment strategies.

Metabolism plays a key role in the maintenance of normal hematopoietic stem cells (HSC) and in the development of leukemia. A better understanding of the metabolic characteristics and dependencies of preleukemic cells could help identify potential therapeutic targets to prevent leukemic transformation. As AML1-ETO, one of the most frequent fusion proteins in acute myeloid leukemia that is encoded by a RUNX1::RUNX1T1 fusion gene, is capable of generating preleukemic clones, in this study, we used a conditional Runx1::Runx1t1 knockin mouse model to evaluate preleukemic cell metabolism. AML1-ETO expression resulted in impaired hematopoietic reconstitution and increased self-renewal ability. Oxidative phosphorylation and glycolysis decreased significantly in these preleukemic cells accompanied by increased HSC quiescence and reduced cell cycling. Furthermore, HSCs expressing AML1-ETO exhibited an increased requirement for fatty acids through metabolic flux. Dietary lipid deprivation or loss of the fatty acid transporter FATP3 by targeted deletion using CRISPR/Cas9 partially restored differentiation. These findings reveal the unique metabolic profile of preleukemic cells and propose FATP3 as a potential target for disrupting leukemogenesis. Significance: Fatty acid metabolism is required for maintenance of preleukemic cells but dispensable for normal hematopoiesis, indicating that dietary lipid deprivation or inhibiting fatty acid uptake may serve as potential strategies to prevent leukemogenesis.

AbstractBesides chemotherapy and hematopoietic stem cell transplantation (HSCT), autologous T cells can also serve as a new treatment approach for AML patients. However, the features of tumor-reactive T cells and their distinctive markers still lack full description. To evaluate the characteristics of tumor-reactive T cells, we collected bone marrow (BM) T cells from newly diagnosed AML patients with RUNX1::RUNX1T1 as examples for paired single-cell RNA sequencing and single-cell V(D)J sequencing. Based on the STARTRAC-like algorithm, we defined bystander T cells and tumor-reactive T cells. Compared with bystander T cells, tumor-reactive T cells presented as senescent-like cytotoxic terminally differentiated T cells (Temra) with upregulated NK-related markers. Additionally, we found ADGRG1 could serve as the specific marker of CD8+ T tumor-reactive T cell and validated it through the Runx1Runx1t1/+; Mx1-Cre mouse model. In chimeric antigen receptor (CAR)-T and target cell system, ADGRG1 was selectively upregulated upon antigen-TCR encounter. Moreover, ADGRG1+CD8+ T cells released a higher level of IFN-γ and showed higher cell-killing ability when exposed to matched AML blasts. Together, our findings depict the single-cell profile of tumor-reactive T cells in AML BM and propose that ADGRG1 can act as an indicator of T cell tumor reactivity in AML, which may be further harnessed for adoptive cell therapy and tumor-reactive TCR enrichment.

Generation of chimeric antigen receptor macrophages (CAR-Ms) from human pluripotent stem cells (hPSCs) offers new prospects for cancer immunotherapy but is currently challenged by low differentiation efficiency and limited function. Here, we develop a highly efficient monolayer-based system that can produce around 6,000 macrophages from a single hPSC within 3 weeks. Based on CAR structure screening, we generate hPSC-CAR-Ms with stable CAR expression and potent tumoricidal activity in vitro. To overcome the loss of tumoricidal activity of hPSC-CAR-Ms in vivo, we use interferon-γ and monophosphoryl lipid A to activate an innate immune response that repolarizes the hPSC-CAR-Ms to tumoricidal macrophages. Moreover, through combined activation of T cells by hPSC-CAR-Ms, we demonstrate that activating a collaborative innate-adaptive immune response can further enhance the anti-tumor effect of hPSC-CAR-Ms in vivo. Collectively, our study provides feasible methodologies that significantly improve the production and function of hPSC-CAR-Ms to support their translation into clinical applications.

Chimeric antigen receptor T (CAR-T) cell therapy initiates new methods and turns the scale of clinical treatment on relapsed/refractory acute T lymphoblastic leukemia (T-ALL). In this study, we generated the second-generation CD7-targeting CAR-T cells with a new antigen-binding single-chain variable fragment sequence and made it universal via CRISPR-based knockout of TRAC and CD7 genes (termed UCAR-T). The CD7 UCAR-T cells can efficiently proliferate and lyse T-ALL tumor cell in vitro, along with prominent proinflammatory cytokines secretion. A Jurkat-based xenograft mouse model further verified the superior cytotoxicity of the UCAR-T cells in vivo. During the UCAR-T construction, we observed a CD4/CD8 ratio shift among CD7-/- T/CAR-T cells, which motivated us to further analyze the effects of CD7 antigen on T/CAR-T cells. We sorted out CD7+/- T or anti-CD19 CAR-T cells after partially CD7 knockout and performed functional, phenotypic detection, as well as translational analysis. CD7-/- CAR-T cells tended to be CD8 negative and showed slightly better cytotoxicity at long-term assay. RNA-seq further confirmed an elevation of activated CD4 memory cell subpopulation. However, limited distinction on crucial regulatory genes and pathways was revealed, suggesting the safety and feasibility of UCAR-T application as well as the potential translational rather than transcriptional regulation of CD7 antigen.

Abstract Background T cell-redirecting bispecific antibodies establish a connection between endogenous T cells and tumor cells, activating T cells function to eliminate tumor cells without ex vivo genetic alteration or manipulation. Here, we developed a novel dual-specific antibody (DuAb) and an enhanced DuAb (EDuAb) with different stimulation signal to activate T cells, and evaluated their impact on the treatment of acute lymphoblastic leukemia (ALL). Methods The expression plasmids of the DuAb and EDuAb containing CD80 molecule were constructed by cloning heavy chain and light chain variable fragments from anti-human CD19 (HI19a) and CD3 (HIT3a) monoclonal antibody hybridomas, respectively. The activation and the anti-tumor efficacy of human T cells mediated by DuAb and EDuAb were evaluated in vitro. B-cell ALL xenograft NSG mouse model was established to investigate the therapeutic effect in vivo. Results EDuAb promoted the optimal expansion of primary human T cells with low expression of inhibitory markers in vitro than DuAb did. Both DuAb and EDuAb showed a similar capability in inducing healthy donor T cells to specifically eliminate B-ALL cell lines and primary blasts from patients. The similar ability was also observed in the patient-derived T cells. In vivo study showed that both DuAb and EDuAb significantly alleviated tumor burden and extended survival of B-ALL xenograft NSG mice. The median survival of PBS, DuAb and EDuAb treatment groups were 27, 38 and 45 days, respectively. The phenotype of T cells and cytokine release in peripheral blood (PB) of B-ALL xenograft NSG mice on day 24 were analyzed as well. The results showed that the proportion of CD8+ T cells and cytokine levels, including IL-2, IFN-γ and TNF-α, were higher in the EDuAb group than that of DuAb. Moreover, both DuAb and EDuAb significantly decreased the residual leukemia cells in PB of B-ALL xenograft NSG mice. Conclusions Both DuAb and EDuAb showed great potential as novel treatments for B-ALL in clinical applications. However, compared to DuAb, EDuAb showed a significant advantage in promoting the proliferation and survival of T cells. Furthermore, EDuAb showed a better promising effect on eliminating tumor cells and extending survival in vivo, which provides new insights for the development of new multi-specific antibodies.

Dear Editor, Antigen escape is responsible for resistance [1] or disease relapse [2] from single-target chimeric antigen receptor (CAR)-T therapy. Dual-target CAR-T therapy has the potential to overcome the escape problem. However, the efficacy and safety assessment of dual-target CAR-T therapy in treating acute myeloid leukemia (AML) need further investigation. Tandem CAR-T therapy has been widely used in research and clinic. However, clustering of two connected single-chain variable fragments (scFvs) [3] and the inappropriate conjugation distance [4] pose a risk of damaging tandem CAR-T cells’ function. Moreover, the bicistronic approach has been proven to be more efficacious than tandem and pooled approaches in treating multiple myeloma [5]. Both CD33 and CD123 are regarded as ideal AML targets, and simultaneously targeting CD33 and CD123 can treat almost all AML patients. Therefore, we developed a CD123×CD33 bicistronic CAR (123×33 biCAR) whose scFvs differed from any existing one to improve the clinical efficacy and to explore its hematotoxicity (Supplementary Materials and Methods), as the only published preclinical study of 123×33 biCAR, designed with different scFvs and vectors, did not assess its safety [6]. The 123×33 biCAR-T cell was designed to cope with the antigen escape problem by expressing fully functional anti-CD123 CAR (123CAR) and anti-CD33 CAR (33CAR) in one T cell (Figure 1A). Two second-generation CARs containing 4-1BB/CD3-ζ intracellular signaling components were linked by T2A sequence and cloned into a lentiviral backbone to obtain the bicistronic CAR vector (Figure 1B). CD123 scFv and CD33 scFv were derived from clone 13C3 and clone HI33a, respectively, both of which were established by our laboratory. Flow cytometry (FCM) analysis showed that both 123CAR and 33CAR were highly expressed (above 40.0%) on T cell surface (Figure 1C). Structure and biological properties of 123×33 biCAR-T. (A) Antigen losing or switching leads to tumor escape, and 123×33 biCAR-T cells overcome this issue by specifically targeting both antigens simultaneously. (B) Schematic diagram of the 123×33 biCAR. 13C3 scFv was incorporated with the 4-1BB/CD3ζ signaling domain to generate a second-generation CAR. CD123 and CD33 CARs were linked via a T2A sequence and cloned into a pCDH vector. (C) CAR expression was detected using FCM. Human recombinant CD123 and CD33 proteins were used to detect the expression of each CAR. F(ab′)2 positive cells represent CAR+ cells. (D) Representative histograms depicting the proportion of the residual target cells Molm13, Jurkat33, Jurkat123 and K562 after co-culture with CAR-T cells at an E:T ratio of 1:1 for 24 h. (n = 3; mean ± SEM; one-way ANOVA with Dunnett’s multiple comparison test; ns, no significance; ***P < 0.001). (E) BLI of luciferase activity in NSG mice on days 3 and 12 after inoculation of Molm13 cells (n = 6 mice per group, one from the 123×33 biCAR group died of anesthesia on day 3). (F) Kaplan–Meier survival curves for the overall survival of the Molm13-inoculated mice (n = 11; log-rank test; *** P < 0.001). Survival observation was conducted independently from BLI to avoid the negative effect of anesthetic injection on mice survival. (G) BLI of Jurkat123- and Jurkat33-inoculated mice from the different treatment groups (n = 4 mice per group) on the indicated days. (H) Kaplan–Meier survival curves for the overall survival of the Jurkat123- and Jurkat33-inoculated mice (n = 7 in each CAR-T group, and n = 6 in the normal T group; log-rank test; *P < 0.05; ***P < 0.001). Survival observation was conducted independently from BLI to avoid the negative effect of anesthetic injection on mice survival. (I-K) scRNA-seq analysis of residual CD34+ cells treated with different T/CAR-T cells. (I) UMAP visualization indicating 0 –17 clusters identified based on single-cell transcriptomes. Cell type was annotated on the corresponding cluster. Each dot represents a single cell, and colors indicate cell clusters. (J) Stacked bar plots indicating the frequencies of defined cell types in different samples. Hematopoietic cells are marked in various tones of red, and T cells are shown in different tones of blue. (K) Dot plot of GSEA results of HSC in different CAR-T groups compared with HSC in normal T group based on the indicated gene sets. (L-M) CFU assay to determine the colony-forming ability of residual CD34+ cells of different groups. (L) Representative photos of colonies in different groups. (M) Histogram showing the colony numbers of each group. (n = 3; mean ± SEM; P values were calculated by two-way ANOVA with Dunnett's multiple comparison test and are shown in the table below the graphs; ns, no significance; *** P < 0.001). Abbreviations: CAR, chimeric antigen receptor; 123×33 biCAR, CD123×CD33 bicistronic CAR; scFvs, single chain variable fragments; FCM, flowcytometry; E:T, effector cell: target cell; SEM, Standard Error of the Mean; Analysis of Variance (ANOVA); BLI, bioluminescent imaging; NSG, nonobese diabetic/severe combined immunodeficiency /IL2Rγ-/-; scRNA-seq, single-cell RNA-sequencing; UMAP, uniform manifold approximation and projection; Teff, effector T cells; Tex, exhausted T cells; Tpro, proliferating T cells; Tact, activated T cells; Tn/Tcm, naïve T/central memory T cells; GSEA, gene set enrichment analysis; HSC, hematopoietic stem cell; CFU, colony forming units; CFU-GEMM, CFU-granulocyte, erythrocyte, macrophage, megakaryocyte; CFU-GM, CFU-granulocyte, macrophage; CFU-M, CFU- macrophage; CFU-G, CFU-granulocyte; BFU-E, burst-forming unit-erythroid. Co-culture test showed that 123×33 biCAR-T cells lysed not only CD123+CD33+ Molm13 cells but also CD123−CD33+ Jurkat (Jurkat33) or CD123+CD33− Jurkat (Jurkat123) cells; in comparison, 123CAR-T or 33CAR-T cells did not lyse Jurkat cells without corresponding antigen; none of three types of CAR-T cells exhibited cytotoxicity toward CD123−CD33−K562 cells (Figure 1D, Supplementary Figures S1-S2). Enzyme-linked immunosorbent assay (ELISA) test showed that 123×33 biCAR-T cells released high levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) against Molm13, Jurkat123 and Jurkat33 cell lines, which were hundreds to thousands folds of that secreted by normal T cells (Supplementary Figure S3). The above data indicated that 123×33 biCAR-T cells have specific cytotoxicity against either CD123 or CD33 in vitro. Two xenograft mouse models were established to evaluate the in vivo efficacy of 123×33 biCAR-T cells. Firstly, a Molm13-NOD/SCID/IL2Rγ-/- (NSG) model was established (Supplementary Figure S4) to mimic the most common situations in AML patients, where CD123 and CD33 are usually expressed concomitantly. The 123×33 biCAR-T performed as well as or better than single-target CAR-T in eradicating Molm13 cells (Figure 1E, Supplementary Figure S5) and prolonging the Molm13-NSG mice survival time (Figure 1F). In addition, an antigen escape model was established by engrafting NSG mice with a mixture of equal number of Jurkat123 and Jurkat33 cells (Supplementary Figure S6). In this Jurkat hybrid model, 123×33 biCAR-T cells showed obvious superiority over the two types of single-target CAR-T cells in suppressing tumor burden (Figure 1G, Supplementary Figure S7) and prolonging survival time (Figure 1H), substantiating the advantage of dual-target CAR in antigen-loss situations. The two main possible reasons for the non-durable anti-leukemia effect in vivo in our study are the rejection of mice against human T cells and limited amount of CAR-T cells in vivo, which was lower than the number reported in the other literature [7]. All things considered, 123×33 biCAR-T cells showed robust anti-AML effect and promising anti-escape capacity in vivo. The off-target cytotoxicity of 123CAR and 33CAR on normal hematopoietic stem and progenitor cells (HSPCs) has been controversial [7-9]. Herein, we first evaluated the influence of 123×33 biCAR on hematopoiesis by several methods. Cord blood-derived CD34+ cells were co-cultured with normal T or CAR-T cells in a 1:1 ratio for 24 h. Although enhanced cytokine secretion (Supplementary Figure S8) was observed in CAR-T groups, none of the three types of CAR-T cells induced a significant reduction in CD34+ cell counts (Supplementary Figure S9) compared to normal T cells, which indicated that CAR-T cells in this study had no obvious specific cytotoxicity against CD34+ cells in vitro. Additionally, single-cell RNA-sequencing (scRNA-seq) analysis was performed to better understand the effect of CAR-T cells on HSPCs. Reduced dimension analysis using Uniform Manifold Approximation and Projection (UMAP) visualization showed 18 distinct clusters (Figure 1I). The cell type was annotated in each cluster in the UMAP plot, and five clusters located in the lower part of the plot were identified as HSPCs: hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), megakaryocyte-erythroid progenitors (MEPs), granulocyte-monocyte progenitors (GMPs), and multi-lymphoid progenitors (MLPs). HSCs and MPPs (Figure 1I-J, Supplementary Figure S10) constituted the majority of HSPCs. The proportion of the HSC subpopulation barely changed among the groups (12.8%, 10.8%, 11.5% and 11.7% in normal T, 123CAR-T, 33CAR-T and 123×33 biCAR-T groups, respectively), while the MPP subpopulation was reduced in CAR-T groups (12.7%, 4.9%, 7.6% and 5.9% in normal T, 123CAR-T, 33CAR-T and 123×33 biCAR-T groups, respectively) (Figure 1J). Therefore, CAR-T cells reduced progenitor cells, but not stem cells, in vitro. The results of Gene Set Enrichment Analysis (GSEA) revealed that the enrichment of quiescence, cell cycle, G2M checkpoint and E2F target-related genes was not altered in the HSC subpopulation by CAR-T cells (Figure 1K, Supplementary Figure S11 Supplementary Table S3). In addition, downregulation of the transforming growth factor (TGF)-β signaling pathway or enrichment of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K-AKT-mTOR) signaling pathway in the HSC subpopulation treated with CAR-T cells was not observed (Figure 1K, Supplementary Figure S11). Taken together, GSEA indicated that 123×33 biCAR-T cells did not significantly alter the stemness of the HSC cluster. Colony forming unit (CFU) assays showed similar amount of hematopoietic colonies grown after three types of CAR-T treatment, which were lower than that in the normal T group (Figure 1L-M). It corroborated the results of scRNA-seq and suggested that alive HSPCs on exposure to CAR-T cells targeting CD123 and/or CD33 can differentiate into multiple lineages of blood cells. Most researchers hold that CD123 [8] or CD33 single-target CAR-T therapy [9] has low toxicity against hematopoiesis, while a minority hold the opposite view [7]. Our results suggested that CAR-T therapies targeting CD123 and/or CD33 were not myeloablative, and scRNA-seq proved that HSCs were exempted and thus probably retained much hematopoietic capacity. Considering the CAR design was not peculiar to our study, the novel scFvs might be the critical factor of the safety profile we observed. Similar findings of Mardiros et al. [8] support this hypothesis, and further studies are needed to explore the mechanism via which scFvs impact the safety of CAR-T cells. Moreover, the concomitant expression of CD123 and CD33 on HSPCs [10] might be one reason why 123×33 biCAR-T cells bring no additional hematotoxicity compared to CD123 or CD33 single-target CAR-T cells. In conclusion, we constructed a novel 123×33 biCAR-T therapy that has great potential to reduce failure or relapse from single-target CAR-T therapy and proved that it has no more hematotoxicity than CD123 or CD33 single-target CAR-T therapy, which provides more data reference for the preclinical and clinical research of CAR-T therapy targeting both CD123 and CD33. Zhenzhen Wang designed and optimized the experiments, analyzed the data, and wrote the manuscript. Yang Lu designed and performed the antibody development and screening experiments and analyzed the related data. Yu Liu designed and operated the supplementary experiment to test cytotoxicity of CAR-T cells on CD34+ cells. Junli Mou performed the animal experiments, CFU test, and single-cell sample preparation. Xiaoyu Liu and Manling Chen contributed to animal experiments. Shaowei Qiu, Bing Wang, and Wei Qi analyzed the scRNA-seq data. YingXi Xu, Qing Rao, Haiyan Xing, Kejing Tang, Zheng Tian and Ying Wang contributed to the discussion of project results and provided feedback on the manuscript. Jianxiang Wang, Dongsheng Xiong, and Shaowei Qiu conceptualized the study. Jianxiang Wang, Dongsheng Xiong, Shaowei Qiu and Min Wang assisted with the review and editing of the manuscript and supervised the study and manuscript preparation. All authors have reviewed the final version of the manuscript. We would like to thank Dr. Shuo Zhang (State Key Laboratory of Experimental Hematology, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China) for her help and training in colony forming units related experiments. The authors declare no conflict of interest. This work was supported by the National Key Research and Development Program of China (2021YFC2500300), the National Natural Science Foundation of China (81830005), Haihe Laboratory of Cell Ecosystem Innovation Fund (HH22KYZX0032) and the Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Sciences (2020-I2M-C&T-A-019). The study was conducted according to the Declaration of Helsinki and was approved by the ethical advisory board of the Institute of Hematology and Blood Diseases Hospital and Ethics Committee of Haihe Laboratory of Cell Ecosystem. (permit number: HHL2022009-EC-1). Written informed consent was obtained from all the participants. All animal experiments were approved in accordance with the guidelines of the Ethics Committee of Haihe Laboratory of Cell Ecosystem (permit number: HHL2022009-EC-1). Not applicable. Original data will be available via email to the corresponding author at [email protected]. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA-Human: HRA004513) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa-human. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Objectives: The translocation between chromosome 8 and 21, resulting in the AML1-ETO fusion gene, is one of the most common cytogenetic abnormalities observed in acute myeloid leukemia (AML). However, AML1-ETO alone is insufficient to cause leukemia, additional secondary events are required for leukemogenesis. CCND2 gene, a key member of the CyclinD2-CDK4/6 complex, plays a crucial role as a cell cycle regulator. In hematopoietic malignancies, CCND2 mutations (particularly with a conserved mutated hotspot on Thr280) have been found in AML, particularly in core binding factor leukemias. The mutated CCND2 protein exhibits greater stability and resistance to proteasomal degradation. Previous research had shown that CCND2 was a transcriptional target of AML1-ETO, indicating that AML1-ETO can promote CCND2 expression. However, it remains unclear whether mutated CCND2 can cooperate with the AML1-ETO to drive leukemia initiation and progression. Methods: In our previous study, a conditional AML1-ETO knock-in mouse model was developed, in which the AML1-ETO fusion gene was tagged with m-Cherry. To investigate the role of CCND2 in this model, the coding sequences of both CCND2 wt (wild-type) and CCND2 mut (mutant with Thr280Ala substitution) were cloned into the retroviral vector MSCV-IRES-EGFP. Subsequently, the fetal liver cells (embryonic day=12.5) from AML1-ETO mice were infected with the corresponding retroviruses. Results: Four months post AML1-ETO induction, the percentage of m-Cherry + GFP + cells of the mice in CCND2 wt and CCND2 mut groups began to increase. The results revealed a substantial upregulation of CCND2 mRNA and protein expression in these groups compared to that of the Vec group. Additionally, the CCND2 mut group exhibited even higher CCND2 protein expression due to impaired protein degradation. Moreover, the shortened survival time and pathological findings in mice from the CCND2 wt and CCND2 mut groups confirmed that they were succumbed to leukemia infiltration. To assess the stemness of leukemia stem cells (LSCs), extreme limiting dilution analysis (ELDA) was conducted, and it showed a significant increase in LSC frequency in the CCND2 wt and CCND2 mut groups compared to that of the Vec group. Notably, transplanting AML1-ETO cells alone didn't confer any competitive advantage in sublethally irradiated mice, while the overexpression of CCND2 provided them with the ability to survive. Transcriptome analysis indicated that compared to the Vec group, CCND2 wt and CCND2 mut groups exhibited notable enrichment in OXPHOS, E2F, and G2M gene sets. Interestingly, the mTORC1 signaling pathway was particularly enriched in the CCND2 mut group compared to the other two groups. To verify this speculation, phosphorylated S6 and total S6 protein levels were assessed through Western blotting. The results showed that compared to Vec group, the p-S6/total S6 ratio show a gradual increase in the CCND2 wt and CCND2 mut groups, indicating activation of the mTOR pathway by CCND2 protein. Everolimus, a selective inhibitor of mTOR, was administered to CCND2 wt and CCND2 mut mice at a dose of 5mg/kg via oral gavage (three times a week) for three consecutive weeks. The Everolimus-treated mice exhibited reduced leukemic burden and fewer number of LSCs compared to that of the vehicle-treated group. Additionally, Everolimus restored the non-leukemic cells and extended their survival time in CCND2 wt and CCND2 mut group mice. Conclusion: In our study, a leukemia mouse model with co-expression of AML1-ETO and CCND2 mutation was successfully established, which demonstrated that the introduction of the CCND2 gene into the existing AML1-ETO model can trigger the development of leukemia. It was also confirmed that CCND2 overexpression results in the upregulation of the mTOR pathway, which drove the progression of leukemia. Everolimus effectively improved the outcome of mice with CCND2 wt or CCND2 mut, suggesting its potential as a therapeutic agent for this subtype of leukemia.

Background aims: Decades after the identification of natural killer (NK) cells as potential effector cells against malignantly transformed cells, an increasing amount of research suggests that NK cells are a prospective choice of immunocytes for cancer immunotherapy in addition to T lymphocytes for cancer immunotherapy. Recent studies have led to a breakthrough in the combination of hematopoietic stem-cell transplantation with allogeneic NK cells infusion for the treatment of malignant tumors. However, the short lifespan of NK cells in patients is the major impediment, limiting their efficacy. Therefore, prolonging the survival of NK cells will promote the application of NK-cell immunotherapy. As we have known, NK cells use a "missing-self" mechanism to lyse target cells and exert their functions through a wide array of activating, co-stimulatory and inhibitory receptors. Our previous study has suggested that CD244 (2B4), one of the co-stimulatory receptors, can improve the function of chimeric antigen receptor NK cells. However, the underlying mechanism of how 2B4 engages in the function of NK cells requires further investigation. Overall, we established a feeder cell with the expression of CD48, the ligand of 2B4, to investigate the function of 2B4-CD48 axis in NK cells, and meanwhile, to explore whether the newly generated feeder cell can improve the function of ex vivo-expanded NK cells.Methods: First, K562 cells overexpressing 4-1BBL and membrane-bound IL-21 (mbIL-21) were constructed (K562-41BBL-mbIL-21) and were sorted to generate the single clone. These widely used feeder cells (K56241BBL-mbIL-21) were named as Basic Feeder hereinafter. Based on the Basic feeder, CD48 was overexpressed and named as CD48 Feeder. Then, the genetically modified feeder cells were used to expand primary NK cells from peripheral blood or umbilical cord blood. In vitro experiments were performed to compare proliferation ability, cytotoxicity, survival and activation/inhibition phenotypes of NK cells stimulated via different feeder cells. K562 cells were injected into nude mice subcutaneously with tail vein injection of NK cells from different feeder system for the detection of NK in vivo persistence and function.Results: Compared with Basic Feeders, CD48 Feeders can promote the proliferation of primary NK cells from peripheral blood and umbilical cord blood and reduce NK cell apoptosis by activating the p-ERK/BCL2 pathway both in vitro and in vivo without affecting overall phenotypes. Furthermore, NK cells expanded via CD48 Feeders showed stronger antitumor capability and infiltration ability into the tumor microenvironment.Conclusions: In this preclinical study, the engagement of the 2B4-CD48 axis can inhibit the apoptosis of NK cells through the p-ERK/BCL2 signal pathway, leading to an improvement in therapeutic efficiency.(c) 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.

Acute myeloid leukemia (AML) is a biologically and clinically heterogeneous disease with a dismal prognosis and limited treatment options. Chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B cell malignancies but a dismal consequences in AML. In our previous study, we found that interleukin-10 receptor (IL-10R) was overexpressed in most AML cells, and played an important role in promoting the stemness of leukemia cells. In this study, we developed a novel ligand-based CAR-T cell targeting IL-10R, which displayed striking cytotoxicity both in vitro and in vivo against AML cells. Except for monocytes, it had no significant adverse effects on the normal hematopoietic system, including CD34+ hematopoietic stem and progenitor cells (HSPCs). In addition, even though the incorporation of IL-10 in the CAR cassette led to phenotypes change, it had few adverse effects on the survival and biological activity of IL-10 CAR-T cells and did not cause excessive proliferation of leukemia cells. Therefore, we propose IL-10R is a novel promising therapeutic candidate for AML, and IL-10R targeted CAR-T therapy provides a new treatment strategy to improve the prognosis of AML.

AML1-ETO fusion gene is the most common cytogenetic subtype of AML, which occurs in approximately 15% of acute myeloid leukemia (AML) patients. More than 85% of patients could achieve complete remission after induction chemotherapy. However, it was less effective on the patients over the age of 60 who cannot tolerate standard chemotherapy. And nearly 30% of patients relapsed eventually with a dismal outcome. Therefore, identification of the initiating abnormal cells to inhibit the early occurrence induced by AML1-ETO is an urgent problem. In this study, an inducible conditional AML1-ETO knock-in murine model (Aml1Eto/+; Mx1-Cre) was successfully established, hereafter called AEKI. The stage of malignant transformation in hematopoietic stem and progenitor cells (HSPC) was identified. We found that the absolute number of LT-HSCs (1.43×106 vs 4.85×103, p<0.0001), ST-HSCs (7.90×106 vs 1.88×104, p<0.0001) and MPPs (6.07×105 vs 1.82×104, p=0.0358) in bone marrow (BM) increased significantly in AEKI group compared with that of control group, while CMPs (8.13×103 vs 1.83×105, p=0.0029), GMPs (8.48×103 vs 3.12×105, p=0.0026) and MEPs (1.25×104 vs 2.03×105, p=0.001) decreased. It suggested that the activation of AML1-ETO in vivo resulted in excessive accumulation of HSCs and differentiation blockage from HSCs to HPCs. However, AML1-ETO alone was insufficient to induce the onset of overt leukemia after 300-day observation of survival. Furthermore, the characteristics of AEKI HSPCs in the pre-leukemic stage were investigated. Colony forming assay (CFC) was performed to evaluate the stemness of HSCs in vitro. The equivalent of 2×103 LT-HSCs were isolated from BM of each group and plated for 14 days. AEKI LT-HSCs gave rise to dysplastic colonies and lost replating capability compared to that of control group (colony counts: 19.33 vs 30.17, p=0.016). Moreover, a progressive decrease in PB chimerism was observed in AEKI recipients via competitive transplantation assay in vivo (15.8% vs 62.5%, p<0.0001), for which BM cellularity exhibited a significant decrease (7.24×106 vs 4.81×107, p<0.0001). But it led to an notably expansion in HSC number (LT-HSC:1.18×105 v 8.59×103, p=0.0035) which had a same tendency as the phenotype of primary mice. Based on Ki67 and DAPI staining, proportion of HSCs in G1 phase increased (59.85% vs 38.63%, p=0.0007), while that in G0 (33.58% vs 44.2%, p=0.045) and S/G2/M (4.86% vs 13.41%, p=0.0002) phases decreased. These results implied that AML1-ETO induction could reduce the quiescence and self-renewal ability of HSCs obviously in comparison with normal HSCs. Bulk RNA sequencing was performed in LT-HSCs and GMPs sorted from both groups. Genesets related to cell proliferation, cell cycle regulation and Myc targets were enriched in LT-HSCs from control group. It was also found that genesets of tricarboxylic acid (TCA) cycle, glycolysis and fatty acid metabolism were enriched in LT-HSCs from control group. To validate it, Seahorse extracellular flux assay was performed to evaluate oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) on HSPC population (c-kitenriched cells). We found that OCR and ECAR both decreased significantly in AEKI group that supported the results from RNA-seq. RNA-seq data also showed that fatty acid metabolic pathway was enriched in AEKI group during the process from LT-HSCs to GMPs, which suggested that AEKI HSPCs maintained abnormal function through excessive fatty acid supply while it was not necessary during normal hematopoiesis. To confirm the above speculation, the mice were fed with fat-free or normal food after the AML-ETO induction in vivo to evaluate the impact of fatty acid on hematopoiesis. Interestingly, BM chimerism increased on the fat-free group (31.56% vs 15.68%, p=0.013), with the absolute number of LT-HSCs (5.16×105 vs 1.80×106, p=0.068), ST-HSCs (1.28×106 vs 2.72×106, p=0.026) and MPPs (4.70×104 vs 2.53×105, p=0.013) reduced obviously compared with in a normal diet. It suggested the excessive accumulation of HSCs was partly recovered. In summary, our study suggested that AML1-ETO induction could cause HSCs to lose self-renewal potential and lead to differentiation disruption during the pre-leukemic stage of AML. Limiting exogenous lipid uptake was able to rescue the phenotype of differentiation disrupt of HSPCs partly and it could be a promising therapeutic strategy.

Hematopoietic differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic cell and gene regulatory networks but often generates blood cells that lack natural function. Here, we performed extensive single-cell transcriptomic analyses to map fate choices and gene expression patterns during hematopoietic differentiation of hPSCs and showed that oxidative metabolism was dysregulated during in vitro directed differentiation. Applying hypoxic conditions at the stage of endothelial-to-hematopoietic transition in vitro effectively promoted the development of arterial specification programs that governed the generation of hematopoietic progenitor cells (HPCs) with functional T cell potential. Following engineered expression of the anti-CD19 chimeric antigen receptor, the T cells generated from arterial endothelium-primed HPCs inhibited tumor growth both in vitro and in vivo. Collectively, our study provides benchmark datasets as a resource to further understand the origins of human hematopoiesis and represents an advance in guiding in vitro generation of functional T cells for clinical applications.

Regulatory T cells (Tregs) could maintain the characteristics of stem cells and inhibit the differentiation of normal hematopoietic stem/progenitor cells. Recent studies have shown that Tregs, as an important component of acute myeloid leukemia (AML) microenvironments, can help AML cells to evade immune surveillance. However, their function in directly regulating the stemness of AML cells remains elusive. In this study, the increased stemness of AML cells promoted by Tregs was verified in vitro and in vivo. The cytokines released by Tregs were explored, the highly expressed anti-inflammatory cytokine IL10 was found, which could promote the stemness of AML cells through the activation of PI3K/AKT signal pathway. Moreover, disrupting the IL10/IL10R/PI3K/AKT signal in AML/ETO c-kitmut (A/Ec) leukemia mice could prolong the mice survival and reduce the stemness of A/Ec leukemia cells. Finally, it was confirmed in patient samples that the proportion of Tregs to leukemia stem cells (LSCs) was positively correlated, and in CD34+ primary AML cells, the activation of PI3K/AKT was stronger in patients with high Tregs’ infiltration. After rhIL10 treatment, primary AML cells showed increased activation of PI3K/AKT signaling. Therefore, blocking the interaction between Tregs and AML cells may be a new approach to target LSCs in AML treatment.

BACKGROUND AIMS:Anti-CD19 chimeric antigen receptor (CAR)-modified T cells have shown dramatic cytotoxicity against B-cell malignancies. Currently, autologous T cells are conventionally used to manufacture CAR T cells. Low quality or insufficient quantity of autologous T cells may lead to failure of CAR T preparations. Moreover, CAR T preparation usually takes 1-2 weeks, which is too long for patients with rapid disease progression to successfully infuse CAR T cells. Thus, the development of a ready-to-use CAR immunotherapy strategy is needed. NK-92, a natural killer (NK) cell line derived from an NK lymphoma patient, has been gradually applied as a CAR-modified effector cell. To avoid the potential development of secondary NK lymphoma in patients, large doses of radiation are used to treat NK-92 cells before clinical application, which ensures the safety but reduces the cytotoxicity of NK-92 cells. Therefore, it is crucial to explore a suitable radiation dose that ensures short life span and good cytotoxicity of CAR NK-92 cells. METHODS:NK-92MI, a modified IL-2-independent NK-92 cell line, was used to establish an anti-CD19 CAR NK. The suitable radiation dose of CAR NK was then explored in vitro and validated in vivo, and the specific cytotoxicity of irradiated and unirradiated CAR NK against CD19+ malignant cells was assessed. RESULTS:CAR NK exhibited specific cytotoxicity against CD19+ malignant cells. Irradiation ensured a short life span of CAR NK in vitro and in vivo. Encouragingly, irradiated CAR NK displayed an anti-CD19+ malignancy capacity similar to that of unirradiated CAR NK. CONCLUSIONS:Five Gy is a suitable radiation dose to ensure the safety and effectiveness of CD19 CAR NK-92MI cells.

Hyperlipidemia is a key clinical feature in patients with nephrotic syndrome (NS) that is associated with the incidence of cardiovascular events. Recent studies have suggested that the disorders of triglycerides, gluconeogenesis and liver glucose metabolism are associated with the abnormal transcription of clock genes. However, changes to the circadian rhythm of blood lipids in NS require further exploration, and the effects of NS on the hepatic clock system remain to be elucidated. In the present study, the impaired diurnal rhythm of the hepatic core clock genes (BMAL1, CLOCK, CRY1, CRY2, PER1 and PER2) significantly induced circadian rhythm abnormalities in liver-specific clock-controlled genes (LXR, CYP7A1, SREBP-1, ABCA1, DEC1 and DEC2; all P<0.05), which were significantly associated with the abnormal diurnal rhythms of triglyceride, total cholesterol, aspartate aminotransferase and alanine aminotransferase (all P<0.05) in rats with Adriamycin-induced nephropathy. Furthermore, a protein-protein interaction network was identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses based on the human database was conducted to obtain signaling pathway and correlation prediction analyses of overall human clock and clock-controlled gene correlations. Strong correlations of the aforementioned clock genes were detected (avg. local clustering coefficient, 0.849) which suggested significant enrichment in circadian rhythm signaling. The present results indicated that damage to hepatic clock systems may impact blood lipid circadian rhythm disorders in NS, and offer a starting point for understanding the crosstalk between peripheral organs and peripheral clock systems.