Acute Lymphoblastic Leukemia Proliferation Research Articles

Overexpression of PTPN21 promotes proliferation of EGF-stimulated acute lymphoblastic leukemia cells via the MAPK signaling pathways

ABSTRACT Objectives This study aims to investigate the role of excessive Protein Tyrosine Phosphatase Non-Receptor Type 21 (PTPN21) in the proliferation of Acute Lymphoblastic Leukemia (ALL) cells with EGF stimulation. Methods PTPN21 was overexpressed in ALL cell lines by lentiviral transfection. Apoptosis was assayed by Annexin V/7-AAD staining. The proliferation and cell cycle of EGF-treated ALL cells were assessed by MTT and Ki-67/7-AAD staining respectively. The phosphorylation of Src tyrosine kinase and mediators of distinct MAPK pathways were assessed by Western blot. Results Overexpression of PTPN21 had minimal effect on the apoptosis of ALL cells, but significantly promoted the proliferation and cell cycle progression of ALL cells stimulated with EGF. The activity of Src tyrosine kinase and the MAPK pathways was elevated. Inhibition of MAPK pathways by specific inhibitors mitigated this pro-proliferative effect of excessive PTPN21 on EGF-stimulated ALL cells. Conclusion PTPN21 may facilitate ALL progression by promoting cell proliferation via the Src/MAPK signaling pathways.

The Expression and Activation of the NF-κB Pathway Correlate with Methotrexate Resistance and Cell Proliferation in Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Although its prognosis continually improves with time, a significant proportion of patients still relapse from the disease because of the leukemia's resistance to therapy. Methotrexate (MTX), a folic-acid antagonist, is a chemotherapy agent commonly used against ALL and as an immune-system suppressant for rheumatoid arthritis that presents multiple and complex mechanisms of action and resistance. Previous studies have shown that MTX modulates the nuclear factor kappa B (NF-κB) pathway, an important family of transcription factors involved in inflammation, immunity, cell survival, and proliferation which are frequently hyperactivated in ALL. Using a gene set enrichment analysis of publicly available gene expression data from 161 newly diagnosed pediatric ALL patients, we found the Tumor necrosis factor α (TNF-α) signaling pathway via NF-κB to be the most enriched Cancer Hallmark in MTX-poor-responder patients. A transcriptomic analysis using a panel of ALL cell lines (six B-cell precursor acute lymphoblastic leukemia and seven T-cell acute lymphoblastic leukemia) also identified the same pathway as differentially enriched among MTX-resistant cell lines, as well as in slowly dividing cells. To better understand the crosstalk between NF-κB activity and MTX resistance, we genetically modified the cell lines to express luciferase under an NF-κB-binding-site promoter. We observed that the fold change in NF-κB activity triggered by TNF-α (but not MTX) treatment correlated with MTX resistance and proliferation across the lines. At the individual gene level, NFKB1 expression was directly associated with a poorer clinical response to MTX and with both an increased TNF-α-triggered NF-κB activation and MTX resistance in the cell lines. Despite these results, the pharmacological inhibition (using BAY 11-7082 and parthenolide) or stimulation (using exogenous TNF-α supplementation) of the NF-κB pathway did not alter the MTX resistance of the cell lines significantly, evidencing a complex interplay between MTX and NF-κB in ALL.

The Mechanism of miR-1294 Targeting SOX15 to Regulate Wnt/β-catenin Signaling Pathway and Promote the Proliferation of Acute Lymphoblastic Leukemia Cells in Children

To explore the effect of abnormal miRNA expression on the proliferation of pediatric acute lymphoblastic leukemia (ALL) cells and its related mechanism. 15 children with ALL and 15 healthy subjects were collected from the Second Affiliated Hospital of Hainan Medical University from July 2018 to March 2021. MiRNA sequencing was performed on their bone marrow cells, and validated using qRT-PCR. MiR-1294 and miR-1294-inhibitory molecule (miR-1294-inhibitor) were transfected into Nalm-6 cells, and the proliferation of Nalm-6 cells was detected by CCK-8 and colony formation assays. Western blot and ELISA were used to detect apoptosis of Nalm-6 cells. Biological prediction of miR-1294 was performed to find the target gene, which was verified by luciferase reporter assay. Si-SOX15 was transfected into Nalm-6 cells, Western blot was used to detect the expression of Wnt signaling pathway-related proteins and to verify the effect of si-SOX15 on the proliferation and apoptosis of Nalm-6 cells. Compared with healthy subjects, 22 miRNAs were significantly upregulated in bone marrow cells of ALL patients, of which miR-1294 was the most significantly upregulated. In addition, the expression level of SOX15 gene was significantly reduced in bone marrow cells of ALL patients. Compared with the NC group, the miR-1294 group showed increased protein expression levels of Wnt3a and β-catenin, faster cell proliferation, and more colony-forming units, while caspase-3 protein expression level and cell apoptosis were reduced. Compared with the NC group, the miR-1294-inhibitor group showed reduced protein expression levels of Wnt3a and β-catenin, slower cell proliferation, and fewer colony-forming units, while caspase-3 protein expression level was increased and apoptosis rate was elevated. miR-1294 had a complementary base-pair with the 3'UTR region of SOX15 , and miR-1294 directly targeted SOX15 . The expression of miR-1294 was negatively correlated with SOX15 in ALL cells. Compared with the si-NC group, the si-SOX15 group showed increased protein expression levels of Wnt3a and β-catenin, accelerated cell proliferation, and decreased caspase-3 protein expression level and cell apoptosis rate. MiR-1294 can target and inhibit SOX15 expression, thus activating the Wnt/β-Catenin signaling pathway to promote the proliferation of ALL cells, inhibit cell apoptosis, and ultimately affect the disease progression.

LncRNA VPS9D1-AS1 promotes cell proliferation in acute lymphoblastic leukemia through modulating GPX1 expression by miR-491-5p and miR-214-3p evasion.

Alterations in messenger RNAs (mRNAs) of protein-coding genes can influence the malignant behaviors of acute lymphoblastic leukemia (ALL) cells. According to the prediction from The Cancer Genome Atlas (TCGA) database, we discovered that glutathione peroxidase 1 (GPX1) was up-regulated in acute myeloid leukemia (LAML) tissues, which pushed us to explore the feasible role and its related modulatory mechanism of GPX1 in ALL. In this research, we first proved the high expression of GPX1 in ALL cells compared with normal cells. Functional assays further revealed that the proliferation was obstructed and the apoptosis was facilitated in ALL cells with silenced GPX1. Then, both miR-491-5p and miR-214-3p that were down-regulated in ALL cells were affirmed to target GPX1. Subsequently, VPS9D1 antisense RNA 1 (VPS9D1-AS1) was recognized as the upstream regulator of miR-491-5p-miR-214-3p/GPX1 axis in a competing endogenous RNA (ceRNA) model. Importantly, we proved that VPS9D1-AS1 served as a tumor promoter in ALL through elevating GPX1. In conclusion, VPS9D1-AS1 contributed to ALL cell proliferation through miR-491-5p-miR-214-3p/GPX1 axis, hinting an optional choice for the treatment of ALL.

MiR-137 inhibits cell proliferation in acute lymphoblastic leukemia by targeting JARID1B.

This study aimed to investigate the possible functions of interaction between JARID1B and miR-137 in ALL. The levels of H3K4me3 and H3K4me2 and the expression of JARID1B and miR-137 were analyzed in six ALL cell lines and 30 ALL patients. The effects of miR-137 and JARID1B on cell proliferation and apoptosis were investigated by silencing or promoting the respective genes. The interaction between miR-137 and JARID1B was confirmed by double-luciferase report assay. The histone H3K4 expressions and miR-137 expression were lower in 30 ALL patients and in six ALL cell lines, while the expression of JARID1B was elevated. A negative correlation was observed between JARID1B and miR-137. Over-expression of miR-137 led to decreasing cell proliferation and increasing apoptosis in MOLT-4 and BALL-1 cells. MiR-137 inhibitor up-regulated JARID1B in these two cell lines, while promoted proliferation in BALL-1 cells only. Dual-luciferase report assay suggested that JARID1B was a direct target of miR-137 in ALL cell lines. The expression of miR-137 was declined in ALL, and JARID1B was directly repressed by miR-137. Aberrant JARID1B expression could result in abnormal histone methylation, which might be one cause of ALL.

Culture filtrate ether extracted metabolites from Streptomyces levis ABRIINW111 increased apoptosis and reduced proliferation in acute lymphoblastic leukemia.

Despite the advances in the discovery of various types of anticancer drugs for curing acute lymphoblastic leukemia (ALL), their toxicity and unfavorable side effects remained as big limitations for therapeutical applications. In this regard, natural products such as Streptomyces -derived agents have shown potential applications as anticancer drugs. The present study deals with evaluating the anti-carcinogenic activity of the ether extracted metabolites derived from Streptomyces on nalm-6 and molt-4 ALL cell lines. MTT assay was performed to evaluate the cytotoxicity effect of Streptomyces sp on nalm-6 and molt-4 cell lines. Apoptosis and proliferation were evaluated by Flow cytometry. Quantitative real-time RT-PCR (qRT-PCR) and western blot were performed to investigate the effect of these metabolites on the mRNA and protein expression levels of P53, Bax, and Bcl2. In both cell lines, extracted metabolites significantly inhibited cell growth and increased apoptosis. Although P53, Bax mRNA and protein expressions were increased, Bcl-2 expression decreased in treated cells compared with control. In addition, the G0/G1 arrest of Nalm-6 cells was induced. These findings of this work show that the ether-extracted metabolites from Streptomyces levis ABRIINW111 can be used as an anti-carcinogenic for acute lymphoblastic leukemia cells.

MiR-181a-5p, an inducer of Wnt-signaling, facilitates cell proliferation in acute lymphoblastic leukemia.

Uncontrolled Wnt signaling causes leukemia. Inactivation of Wnt antagonists could play an important role in leukemia progression by activating the Wnt/β-catenin pathway. Wnt inhibitory factor-1 (WIF1) is one of the important Wnt antagonists. Few miRNAs have been reported to directly target this gene in hematopoiesis. Here, we observed that miR-181a-5p expression was markedly overexpressed in several leukemia cell lines and acute lymphoblastic leukemia (ALL) samples compared with that noted in normal peripheral blood mononuclear cells. MTT assays, soft agar colony formation assays and flow cytometry analysis collectively showed that ectopic expression of miR-181a-5p induced ALL cell growth and proliferation. Furthermore, a mechanistic study disclosed that miR-181a-5p directly downregulated WIF1 expression by binding to its 3'-UTR, and further activated Wnt/β‑catenin signaling. These findings provide a novel mechanistic insight into the role of miR-181a-5p in ALL cell growth and proliferation and implicate miR-181a-5p as an attractive candidate for ALL therapy.

Regulation of cellular proliferation in acute lymphoblastic leukemia by Casein Kinase II (CK2) and Ikaros.

The IKZF1 gene encodes the Ikaros protein, a zinc finger transcriptional factor that acts as a master regulator of hematopoiesis and a tumor suppressor in leukemia. Impaired activity of Ikaros is associated with the development of high-risk acute lymphoblastic leukemia (ALL) with a poor prognosis. The molecular mechanisms that regulate Ikaros' function as a tumor suppressor and regulator of cellular proliferation are not well understood. We demonstrated that Ikaros is a substrate for Casein Kinase II (CK2), an oncogenic kinase that is overexpressed in ALL. Phosphorylation of Ikaros by CK2 impairs Ikaros' DNA-binding ability, as well as Ikaros' ability to regulate gene expression and function as a tumor suppressor in leukemia. Targeting CK2 with specific inhibitors restores Ikaros' function as a transcriptional regulator and tumor suppressor resulting in a therapeutic, anti-leukemia effect in a preclinical model of ALL. Here, we review the genes and pathways that are regulated by Ikaros and the molecular mechanisms through which Ikaros and CK2 regulate cellular proliferation in leukemia.

Effects of p38α/β inhibition on acute lymphoblastic leukemia proliferation and survival in vivo.

P38α/β has been described as a tumor-suppressor controlling cell cycle checkpoints and senescence in epithelial malignancies. However, p38α/β also regulates other cellular processes. Here, we describe a role of p38α/β as a regulator of acute lymphoblastic leukemia (ALL) proliferation and survival in experimental ALL models. We also report first evidence that p38α/β phosphorylation is associated with the occurrence of relapses in TEL-AML1-positive leukemia. First, in vitro experiments show that p38α/β signaling is induced in a cyclical manner upon initiation of proliferation and remains activated during log-phase of cell growth. Next, we provide evidence that growth-permissive signals in the bone marrow activate p38α/β in a novel avian ALL model, in which therapeutic targeting can be tested. We further demonstrate that p38α/β inhibition by small molecules can suppress leukemic expansion and prolong survival of mice bearing ALL cell lines and primary cells. Knockdown of p38α strongly delays leukemogenesis in mice xenografted with cell lines. Finally, we show that in xenografted TEL-AML1 patients, ex vivo p38α/β phosphorylation is associated with an inferior long-term relapse-free survival. We propose p38α/β as a mediator of proliferation and survival in ALL and show first preclinical evidence for p38α/β inhibition as an adjunct approach to conventional therapies.

MicroRNA-181a enhances cell proliferation in acute lymphoblastic leukemia by targeting EGR1

Acute lymphoblastic leukemia (ALL) is an aggressive cancer that occurs in both children and adults. Starting from an integrated analysis of miRNA/mRNA expression profiles in 20 ALL patients, we identify a negative correlation between miR-181a and EGR1. Coherently, miR-181a over-expression in Jurkat T-ALL cells decreases EGR1 expression, increasing cell proliferation and enhancing the cell-cycle progression from G1 to S phase. We show that EGR1 is a new direct target of miR-181a. Our findings suggest that miR-181a behaves as an onco-miRNA in ALL by down-regulating EGR1.

Plerixafor (AMD3100) induces prolonged mobilization of acute lymphoblastic leukemia cells and increases the proportion of cycling cells in the blood in mice

The CXCR4 antagonist Plerixafor (AMD3100) induces the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. AMD3100 similarly induces the mobilization of human acute lymphoblastic leukemia (ALL) cells into the blood in mice. In this study, the temporal response of pre-B ALL cells to AMD3100 was compared with that of normal hematopoietic progenitor cells (HPC) using an NOD/SCID xenograft model of ALL and BALB/c mice, respectively. ALL cells remained in the circulation up to 6 hours after AMD3100 administration, by which time normal HPCs were no longer detectable. AMD3100 also increased the proportion of actively cycling ALL cells in the peripheral blood. Together, these data suggest that ALL cells are more sensitive to the effects of bone marrow disruption than normal progenitors. Using the NOD/SCID xenograft model, we demonstrated that AMD3100 increased the efficacy of the cell cycle specific drug vincristine, resulting in reduced disease levels in the blood and spleens of animals over 3 weeks and extended the survival of NOD/SCID mice with ALL. These data demonstrate that mobilizing agents can increase the therapeutic effect of cell cycle dependent chemotherapeutic agents.

Aberrant Expression of BAFF System Molecules on B-Cell Precursor Leukemia Modulates Tumor Cell Survival: Novel Targets for Therapeutic Intervention.

Identification of pathways that support the survival and proliferation of acute lymphoblastic leukemia (ALL) cells will provide novel targets for therapeutic intervention. BAFF and APRIL, with their receptors, BAFF-R, BCMA and TACI are members of the TNF family that play a critical role in the survival of mature B cells but not of normal B cell precursors. We sought to determine whether BAFF/APRIL and their receptors might be aberrantly expressed on B-cell precursor ALL. Surprisingly, tumor cells from 28 of 29 ALL patients expressed mRNA for at least one of these receptors. Using flow cytometric analysis, surface expression of BAFF-R, BCMA, and TACI protein was detected on 89%, 78% and 56% of B-lineage ALL, respectively. Binding assays using a BAFF fusion protein demonstrated that ALL cells expressed high-affinity receptors for BAFF. Stimulation of leukemia cells with BAFF fusion protein triggered the activation of the NFκB pathway with phosphorylation of NFκB2 p100 (Ser864), and its processing to p52. However, BAFF binding did not trigger phosphorylation of IKKα, suggesting that BAFF-mediated activation of NFκB on leukemia cells occurs in an IKK-independent manner. These observations demonstrate that B-cell precursor ALL express functional receptors for BAFF/APRIL ligands. To examine the biological significance of the BAFF system in precursor B-cell ALL, we performed survival and proliferation assays using a myc-tagged BAFF. Our results indicate that while BAFF promoted survival of B-ALL cells, it did not induce their proliferation. Moreover, BAFF binding to leukemia cells was accompanied by increased expression of Bcl-2. To determine the source of BAFF in the leukemic BM, expression of membrane-bound BAFF and APRIL was analyzed in both leukemia cells and cells from the BM microenvironment (BM endothelium, BM mesenchymal stem cells and BM stroma). Surprisingly, leukemia cells expressed BAFF in all patients tested, and APRIL was detected in 94% of the cases. BM microenvironmental cells were found to express membrane-bound BAFF but not APRIL. These findings demonstrate that leukemia B-cell precursors express functional receptors for BAFF system ligands. Importantly, we show that BAFF-mediated stimulation of leukemia cells triggers the activation of the NFκB pathway through an IKK-independent mechanism, and promotes the survival of leukemia cells. The differential expression of BAFF and APRIL on B-ALL cells and the BM microenviromental cells suggests that these ligands may act through both autocrine and juxtacrine mechanisms. Our studies indicate that the BAFF/APRIL ligand-receptor axis might provide new opportunities for therapeutic intervention in B-cell ALL.

Somatostatin and its cyclic octapeptide analog SMS 201–995 as inhibitors of proliferation of human acute lymphoblastic and acute myeloid leukemia

Somatostatin (SS) is a 14 amino acid peptide which is secreted by the hypothalamus and the pancreatic islets. It expresses antiproliferative activity in various organ systems, experiments have suggested effects of SS on hematopoietic cells. Here we present investigations regarding the effect of SS and its analog SMS 201–995 (SMS) on the in vitro proliferation of acute lymphoblastic leukemia (ALL; n = 7 cases), acute myeloid leukemia (AML; n = 21 cases) and chronic lymphocytic leukemia (CLL; n = 2 cases). Both SS and SMS inhibited spontaneous leukemic cell growth in approximately 1 3 of cases (i.e. 7 19 ). G-CSF stimulated AML cells were inhibited by SMS in 11 21 cases. AML cell proliferation induced by IL-3 or GM-CSF was suppressed in only 3 21 and 6 21 cases, respectively. In ALL cells, IL-7-induced proliferation was suppressed by SMS in 3 7 cases. The effect of SMS seemed to depend on the type of the hematopoietic growth factor, and on their concentrations. In fact, high concentrations of G-CSF could override SMS blocking completely. Colony formation by normal marrow progenitors and DNA synthesis by HL-60 and T11 65 leukemic cell lines were not affected by SMS. In conclusion, somatostatin may act as a negative regulator of the proliferative activity of human leukemia.