Slow-replicating leukemia cells represent a leukemia stem cell population with high cell-surface CD74 expression.

Persistence of quiescent leukemia stem cells (LSCs) after treatment most likely contributes to chemotherapy resistance and poor prognosis of leukemia patients. Identification of this quiescent cell population would facilitate eradicating LSCs. Here, using a cell-tracing PKH26 (PKH) dye that can be equally distributed to daughter cells following cell division in vivo, we identify a label-retaining slow-cycling leukemia cell population from AML1-ETO9a (AE9a) leukemic mice. We find that, compared with cells not maintaining PKH-staining, a higher proportion of PKH-retaining cells are in G0 phase, and PKH-retaining cells exhibit increased colony formation ability and leukemia initiation potential. In addition, PKH-retaining cells possess high chemo-resistance and are more likely to be localized to the endosteal bone marrow region. Based on the transcriptional signature, HLA class II histocompatibility antigen gamma chain (Cd74) is highly expressed in PKH-retaining leukemia cells. Furthermore, cell surface CD74 was identified to be highly expressed in LSCs of AE9a mice and CD34+ human leukemia cells. Compared to Lin-CD74- leukemia cells, Lin-CD74+ leukemia cells of AE9a mice exhibit higher stemness properties. Collectively, our findings reveal that the identified slow-cycling leukemia cell population represents an LSC population, and CD74+ leukemia cells possess stemness properties, suggesting that CD74 is a candidate LSC surface marker.

[Bone Marrow Osteoblasts Promotes the Proliferation Leukemia Stem Cell by Up-regulating Interleukin-1].

OBJECTIVE: To explore the extrinsic regulation mechanism of bone marrow microenvironment in leukemia cells, and investigate the promoting effect of osteoblast niche on the proliferation and self-renewal of leukemia stem cell by up-regulating the expression of interleukin-1 (IL-1) in leukemia cell. METHODS: The gene expression profiles on leukemia cells derived from AE9a mouse bone marrow endosteum and central bone marrow were determined by RNA sequencing and gene set enrichment analysis (GSEA). Quantitative real-time PCR (qRT-PCR) was used to detect the expression of IL-1 in AE9a mouse leukemia cells co-cultured with or without osteoblasts in vitro. In addition, qRT-PCR was also used to determine the expression of IL-1 in bone marrow mononuclear cell (BMMNC) from 43 patients with acute myeloid leukemia (AML). For leukemia cells co-cultured with osteoblasts or treated with IL-1ß, colony forming ability of AE9a leukemia cells was determined by colony formation assay. RESULTS: In AE9a leukemia mouse, RNA-seq data and GSEA showed that the enrichment of the upregulated genes in leukemia cells located in endosteum fell into inflammatory response gene set, among them, IL-1α and IL-1ß were significantly higher expressed in AE9a leukemia cells that located osteoblast niche (IL-1α: P<0.001, IL-1ß:P<0.001). After AE9a leukemia cells were co-cultured with osteoblasts in vitro, the expression of IL-1α and IL-1ß in leukemia cells were increased by 2.5 and 3.5 times respectively. In colony formation assay, the number of colonies was increased significantly after leukemia cells were co-cultured with osteoblasts (P<0.001). In addition, when AE9a leukemia cells were treated with IL-1ß, the number of colonies was also increased significantly (P<0.01). In AML patients, BMMNC with high percentage of CD34 positive cells exhibited higher level of IL-1 expression. CONCLUSION: Osteoblast niche can promote leukemia cell proliferation and self-renewal through up-regulating the expression of IL-1 in leukemia cells. In AML patients, the expression level of IL-1 was correlated to the percentage of CD34 positive cells in BMMNC.

Anti-tumor effects of vascular endothelial growth factor/vascular endothelial growth factor receptor binding domain-modified chimeric antigen receptor T cells.

BACKGROUND AIMS: The vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR) signaling pathway plays an important role in angiogenesis and lymphangiogenesis, which are closely related to tumor cell growth, survival, tissue infiltration and metastasis. Blocking/interfering with the interaction between VEGF and VEGFR to inhibit angiogenesis/lymphangiogenesis has become an important means of tumor therapy. METHODS: Here the authors designed a novel chimeric antigen receptor (CAR) lentiviral vector expressing the VEGF-C domain targeting both VEGFR-2 and VEGFR-3 (VEGFR-2/3 CAR) and then transduced CD3-positive T cells with VEGFR-2/3 CAR lentivirus. RESULTS: After co-culturing with target cells, VEGFR-2/3 CAR T cells showed potent cytotoxicity against both VEGFR-2- and VEGFR-3-positive breast cancer cells, with increased simultaneous secretion of interferon gamma, tumor necrosis factor alpha and interleukin-2 cytokines. Moreover, CAR T cells were able to destroy the tubular structures formed by human umbilical vein endothelial cells and significantly inhibit the growth, infiltration and metastasis of orthotopic mammary xenograft tumors in a female BALB/c nude mice model. CONCLUSIONS: The authors' results indicate that VEGFR-2/3 CAR T cells targeting both VEGFR-2 and VEGFR-3 have significant anti-tumor activity, which expands the application of conventional CAR T-cell therapy.

Mutant U2AF1-induced differential alternative splicing causes an oxidative stress in bone marrow stromal cells.

Alternative splicing (AS) is a critical regulatory process of gene expression. In bone marrow microenvironment, AS plays a critical role in mesenchymal stem cells fate determination by forming distinct isoforms of important regulators. As a spliceosome factor, U2AF1 is essential for the catalysis of pre-mRNA splicing, and its mutation can cause differential AS events. In the present study, by forced expression of mutant U2AF1 (U2AF1S34F) in the mouse bone marrow stroma OP9 cells, we determine AS changes in U2AF1S34F transduced OP9 cells and investigate their role in stroma cell biological functions. We find that abundant differential RNA splicing events are induced by U2AF1S34F in OP9 cells. U2AF1S34F causes increased generation of hydrogen peroxide, promotes production of cytokines and chemokines. U2AF1S34F transduced OP9 cells also exhibit dysfunction of mitochondria. RNA-seq data, gene ontology (GO), and gene set enrichment analysis reveal that differentially expressed genes downregulated in response to U2AF1S34F are enriched in peroxisome component and function. U2AF1S34F can also cause release of hydrogen peroxide from OP9 cells. Furthermore, we investigate the influence of U2AF1S34F-induced oxidative stress in stromal cells on hematopoietic cells. When co-culturing mouse bone marrow mononuclear cells with OP9 cells, the U2AF1S34F expressing OP9 cells induce phosphorylation of histone H2AX in hematopoietic cells. Collectively, our results reveal that mutant U2AF1-induced differential AS events cause oxidative stress in bone marrow stromal cells and can further lead to DNA damage and genomic instability in hematopoietic cells.

Mitochondrial dysfunction and oxidative stress in bone marrow stromal cells induced by daunorubicin leads to DNA damage in hematopoietic cells.

Cytotoxic chemotherapies could cause the dysregulation of hematopoiesis and even put patients at increased risk of hematopoietic malignancy. Therapy-related leukemia is mainly caused by cytotoxic chemotherapy-induced genetic mutations in hematopoietic stem/progenitor cells (HSPCs). In addition to the intrinsic mechanism, some extrinsic events occurring in the bone marrow (BM) microenvironment are also possible mechanisms involved in genetic alteration. In the present study, we investigated the damage to BM stromal cells induced by a chemotherapy drug, daunorubicin (DNR) and further identified the DNA damage in hematopoietic cells caused by drug-treated stromal cells. It was found that treatment with DNR in mice caused a temporary reduction in cell number in each BM stromal cell subpopulation and the impairment of clonal growth potential in BM stromal cells. DNR treatment led to a tendency of senescence, generation of intracellular reactive oxygen species, production of cytokines and chemokines, and dysfunction of mitochondrial in stromal cells. Transcriptome microarray data and gene ontology (GO) or gene set enrichment analysis (GSEA) showed that differentially expressed genes that were down-regulated in response to DNR treatment were significantly enriched in mitochondrion function, and negative regulators of reactive oxygen species. Surprisingly, it was found that DNR-treated stromal cells secreted high levels of H2O2 into the culture supernatant. Furthermore, coculture of hematopoietic cells with DNR-treated stromal cells led to the accumulation of DNA damage as determined by the levels of histone H2AX phosphorylation and 8-oxo-2'-deoxyguanosine in hematopoietic cells. Overall, our results suggest that DNR-induced BM stromal cell damage can lead to genomic instability in hematopoietic cells.

[Oxidative Damage of Bone Marrow Stromal Cells Caused by Chemotherapy Drugs].

OBJECTIVE: To explore the oxidative damage of OP9 cells induced by daunorubicin (DNR) treatment. METHODS: The TMRM probe was used to detect mitochondrial membrane potential by flow cytometry; the reactive oxygen species (ROS) was determined by flow cytometry DCFDA probe; the real-time PCR was used to detect the molecular expression of antioxidant enzyme，glutathione peroxidase (GPX) in OP9 cells; the expression of γ-H2AX was determined by flow cytometry. RESULTS: Compared with normal OP9 cells, the positive rate of TMRM in DNR-treated OP9 cells decreased by 56.7% (P＜0.05); the positive rate of DCFDA in DNR-treated OP9 cells increased by 3.52 times (P＜0.01). Compared with normal OP9 cells, DNR-treated OP9 cells showed a decrease in the expression of GPX4 by 44.22% (P＜0.001); the expression of GPX7 decreased by 65.7% (P＜0.001); the expression of GPX8 decreased by 24.7% (P＜0.001); the positive rate of γ-H2AX in DNR-treated OP9 cells increased (P＜0.05). CONCLUSION: After DNR treatment, mitochondrial membrane potential of OP9 cells decreases; the level of reactive oxygen species increases; the expression of glutathione peroxidase (GPX) molecules decreases significantly; genomic instability increases obviously; the oxidative damage of cells increased.