mTOR Inhibition by Everolimus in Childhood Acute Lymphoblastic Leukemia Induces Caspase-Independent Cell Death

Increasingly, anti-cancer medications are being reported to induce cell death mechanisms other than apoptosis. Activating alternate death mechanisms introduces the potential to kill cells that have defects in their apoptotic machinery, as is commonly observed in cancer cells, including in hematological malignancies. We, and others, have previously reported that the mTOR inhibitor everolimus has pre-clinical efficacy and induces caspase-independent cell death in acute lymphoblastic leukemia cells. Furthermore, everolimus is currently in clinical trial for acute lymphoblastic leukemia. Here we characterize the death mechanism activated by everolimus in acute lymphoblastic leukemia cells. We find that cell death is caspase-independent and lacks the morphology associated with apoptosis. Although mitochondrial depolarization is an early event, permeabilization of the outer mitochondrial membrane only occurs after cell death has occurred. While morphological and biochemical evidence shows that autophagy is clearly present it is not responsible for the observed cell death. There are a number of features consistent with paraptosis including morphology, caspase-independence, and the requirement for new protein synthesis. However in contrast to some reports of paraptosis, the activation of JNK signaling was not required for everolimus-induced cell death. Overall in acute lymphoblastic leukemia cells everolimus induces a cell death that resembles paraptosis.     

Sulforaphane Induces Cell Cycle Arrest and Apoptosis in Acute Lymphoblastic Leukemia Cells

Acute lymphoblastic leukemia (ALL) is the most common hematological cancer in children. Although risk-adaptive therapy, CNS-directed chemotherapy, and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs are needed as frontline treatments in high-risk disease and as salvage agents in relapsed ALL. In this study, we report that purified sulforaphane, a natural isothiocyanate found in cruciferous vegetables, has anti-leukemic properties in a broad range of ALL cell lines and primary lymphoblasts from pediatric T-ALL and pre-B ALL patients. The treatment of ALL leukemic cells with sulforaphane resulted in dose-dependent apoptosis and G2/M cell cycle arrest, which was associated with the activation of caspases (3, 8, and 9), inactivation of PARP, p53-independent upregulation of p21^CIP1/WAF1, and inhibition of the Cdc2/Cyclin B1 complex. Interestingly, sulforaphane also inhibited the AKT and mTOR survival pathways in most of the tested cell lines by lowering the levels of both total and phosphorylated proteins. Finally, the administration of sulforaphane to the ALL xenograft models resulted in a reduction of tumor burden, particularly following oral administration, suggesting a potential role as an adjunctive agent to improve the therapeutic response in high-risk ALL patients with activated AKT signaling.     

microRNA在急性淋巴细胞白血病中的作用

MicroRNAs(miRNAs)是一类非编码小RNA分子,其通过降解mRNAs或抑制mRNAs翻译来负调控基因表达并可能调控着几乎每一个细胞生理进程.急性淋巴细胞白血病(ALL)是儿童时期最常见的血液肿瘤,miRNA的异常表达谱对于ALL的发病机制与临床应用有至关重要的作用,因此miRNAs已迅速成为ALL潜在的治疗靶点.     

Molecular Genetics of Pre-B Acute Lymphoblastic Leukemia Sister Cell Lines during Disease Progression

For many years, immortalized tumor cell lines have been used as reliable tools to understand the function of oncogenes and tumor suppressor genes. Today, we know that tumors can comprise subclones with common and with subclone-specific genetic alterations. We sequenced DNA and RNA of sequential sister cell lines obtained from patients with pre-B acute lymphoblastic leukemia at different phases of the disease. All five pairs of cell lines carry alterations that are typical for this disease: loss of tumor suppressors (CDKN2A, CDKN2B), expression of fusion genes (ETV6-RUNX1, BCR-ABL1, MEF2D-BCL9) or of genes targeted by point mutations (KRAS A146T, NRAS G12C, PAX5 R38H). MEF2D-BCL9 and PAX R38H mutations in cell lines have hitherto been undescribed, suggesting that YCUB-4 (MEF2D-BCL9), PC-53 (PAX R38H) and their sister cell lines will be useful models to elucidate the function of these genes. All aberrations mentioned above occur in both sister cell lines, demonstrating that the sisters derive from a common ancestor. However, we also found mutations that are specific for one sister cell line only, pointing to individual subclones of the primary tumor as originating cells. Our data show that sequential sister cell lines can be used to study the clonal development of tumors and to elucidate the function of common and clone-specific mutations.     

High-valency Anti-CD99 Antibodies Toward the Treatment of T Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that currently requires intensive chemotherapy. While childhood T-ALL is associated with high cure rates, adult T-ALL is not, and both are associated with significant short- and long-term morbidities. Thus, less toxic and effective strategies to treat T-ALL are needed. CD99 is overexpressed on T-ALL blasts at diagnosis and at relapse. Although targeting CD99 with cytotoxic antibodies has been proposed, the molecular features required for their activity are undefined. We identified human antibodies that selectively bound to the extracellular domain of human CD99, and the most potent clone, 10A1, shared an epitope with a previously described cytotoxic IgM antibody. We engineered clone 10A1 in bivalent, trivalent, tetravalent, and dodecavalent formats. Increasing the antibody valency beyond two had no effects on binding to T-ALL cells. In contrast, a valency of ≥3 was required for cytotoxicity, suggesting a mechanism of action in which an antibody clusters ≥3 CD99 molecules to induce cytotoxicity. We developed a human IgG-based tetravalent version of 10A1 that exhibited cytotoxic activity to T-ALL cells but not to healthy peripheral blood cells. The crystal structure of the 10A1 Fab in complex with a CD99 fragment revealed that the antibody primarily recognizes a proline-rich motif (PRM) of CD99 in a manner reminiscent of SH3-PRM interactions. This work further validates CD99 as a promising therapeutic target in T-ALL and defines a pathway toward the development of a selective therapy against T-ALL.     

More Related Gene Pathways to Vincristine-Induced Death Events in a Human T-Acute Lymphoblastic Leukemia Cell Line

Background:Acute lymphoblastic leukemia (ALL) is common in children but rare in adults. Vincristine (VCR) is one of the drugs used at the beginning of treatment. Some genes are resistant to VCR in B-ALL.Methods:Here, we examined the effect of VCR on gene expression changes in a T-ALL cell line, Jurkat. The MTT method was used to determine the IC₅₀ in Jurkat cells treated with different concentrations of VCR for 48 and 72 hours. Total RNA was isolated from the cells and cDNA was prepared. The Human Cancer Drug Target PCR Array kit was used to evaluate the 84 gene expression changes in Jurkat cells. Protein-protein interaction was analyzed by STRING software.Results:We identified 66 differentially expressed genes as comparison to untreated cells. The response to VCR-induced apoptotic events was remarkable in the pathways of heat shock protein, topoisomerases, protein kinases, cathepsins and cell cycle. In other pathways, there were resistant genes as well as sensitive genes to VCR treatment. Some proteins like HSP90AA1 and ESR1 had determining associations with other proteins.Conclusion:The results suggest VCR target genes in T-ALL cells may be beneficial biomarkers for ALL treatment and can be used to select appropriate synergistic drugs for VCR.Key Words: ALL, Gene expression profile, Jurkat, PCR array, Vincristine     

The Wnt Pathway Controls Cell Death Engulfment,Spindle Orientation,and Migration through CED-10/Rac

Wnt signalling pathways have extremely diverse functions in animals, including induction of cell fates or tumours, guidance of cell movements during gastrulation, and the induction of cell polarity. Wnt can induce polar changes in cellular morphology by a remodelling of the cytoskeleton. However, how activation of the Frizzled receptor induces cytoskeleton rearrangement is not well understood. We show, by an in depth 4-D microscopy analysis, that the Caenorhabditis elegans Wnt pathway signals to CED-10/Rac via two separate branches to regulate modulation of the cytoskeleton in different cellular situations. Apoptotic cell clearance and migration of the distal tip cell require the MOM-5/Fz receptor, GSK-3 kinase, and APC/APR-1, which activate the CED-2/5/12 branch of the engulfment machinery. MOM-5 (Frizzled) thus can function as an engulfment receptor in C. elegans. Our epistatic analyses also suggest that the two partially redundant signalling pathways defined earlier for engulfment may act in a single pathway in early embryos. By contrast, rearrangement of mitotic spindles requires the MOM-5/Fz receptor, GSK-3 kinase, and β-catenins, but not the downstream factors LIT-1/NLK or POP-1/Tcf. Taken together, our results indicate that in multiple developmental processes, CED-10/Rac can link polar signals mediated by the Wnt pathway to rearrangements of the cytoskeleton.     

Vitamin D Receptor Controls Cell Stemness in Acute Myeloid Leukemia and in Normal Bone Marrow

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Cycloart-24-ene-26-ol-3-one,a New Cycloartane Isolated from Leaves of Aglaia exima Triggers Tumour Necrosis Factor-Receptor 1-Mediated Caspase-Dependent Apoptosis in Colon Cancer Cell Line

Plants in the Meliaceae family are known to possess interesting biological activities, such as antimalaral, antihypertensive and antitumour activities. Previously, our group reported the plant-derived compound cycloart-24-ene-26-ol-3-one isolated from the hexane extracts of Aglaia exima leaves, which shows cytotoxicity towards various cancer cell lines, in particular, colon cancer cell lines. In this report, we further demonstrate that cycloart-24-ene-26-ol-3-one, from here forth known as cycloartane, reduces the viability of the colon cancer cell lines HT-29 and CaCO-2 in a dose- and time-dependent manner. Further elucidation of the compound’s mechanism showed that it binds to tumour necrosis factor-receptor 1 (TNF-R1) leading to the initiation of caspase-8 and, through the activation of Bid, in the activation of caspase-9. This activity causes a reduction in mitochondrial membrane potential (MMP) and the release of cytochrome-C. The activation of caspase-8 and -9 both act to commit the cancer cells to apoptosis through downstream caspase-3/7 activation, PARP cleavage and the lack of NFkB translocation into the nucleus. A molecular docking study showed that the cycloartane binds to the receptor through a hydrophobic interaction with cysteine-96 and hydrogen bonds with lysine-75 and -132. The results show that further development of the cycloartane as an anti-cancer drug is worthwhile.     

Metabolomics of the Tumor Microenvironment in Pediatric Acute Lymphoblastic Leukemia

The tumor microenvironment is emerging as an important therapeutic target. Most studies, however, are focused on the protein components, and relatively little is known of how the microenvironmental metabolome might influence tumor survival. In this study, we examined the metabolic profiles of paired bone marrow (BM) and peripheral blood (PB) samples from 10 children with acute lymphoblastic leukemia (ALL). BM and PB samples from the same patient were collected at the time of diagnosis and after 29 days of induction therapy, at which point all patients were in remission. We employed two analytical platforms, high-resolution magnetic resonance spectroscopy and gas chromatography-mass spectrometry, to identify and quantify 102 metabolites in the BM and PB. Standard ALL therapy, which includes l-asparaginase, completely removed circulating asparagine, but not glutamine. Statistical analyses of metabolite correlations and network reconstructions showed that the untreated BM microenvironment was characterized by a significant network-level signature: a cluster of highly correlated lipids and metabolites involved in lipid metabolism (p<0.006). In contrast, the strongest correlations in the BM upon remission were observed among amino acid metabolites and derivatives (p<9.2×10^-10). This study provides evidence that metabolic characterization of the cancer niche could generate new hypotheses for the development of cancer therapies.     

Volume-Sensitive Chloride Channels are Involved in Maintenance of Basal Cell Volume in Human Acute Lymphoblastic Leukemia Cells

Chloride channels are expressed ubiquitously in different cells. However, the activation and roles of volume-activated chloride channels under normal isotonic conditions are not clarified, especially in lymphatic cells. In this study, the activation of basal and volume-activated chloride currents and their roles in maintenance of basal cell volume under isotonic conditions were investigated in human acute lymphoblastic leukemia Molt4 cells. The patch-clamp technique and time-lapse image analysis were employed to record whole-cell currents and cell volume changes. Under isotonic conditions, a basal chloride current was recorded. The current was weakly outward-rectified and volume-sensitive and was not inactivated obviously in the observation period. A 47% hypertonic bath solution and the chloride channel blockers NPPB and tamoxifen suppressed the current. Exposure of cells to 47% hypotonic bath solution activated further the basal current. The hypotonicity-activated current possessed properties similar to those of the basal current and was inhibited by NPPB, tamoxifen, ATP and hypertonic bath solution. Furthermore, extracellular hypotonic challenges swelled the cells and induced a regulatory volume decrease (RVD). Extracellular applications of NPPB, tamoxifen and ATP swelled the cells under isotonic conditions and inhibited the RVD induced by hypotonic cell swelling. The results suggest that some volume-activated chloride channels are activated under isotonic conditions, resulting in the appearance of the basal chloride current, which plays an important role in the maintenance of basal cell volume in lymphoblastic leukemia cells. Chloride channels can be activated further to induce a regulatory volume recovery when cells are swollen.     

Caspase-1-Dependent and -Independent Cell Death Pathways in Burkholderia pseudomallei Infection of Macrophages

The cytosolic pathogen Burkholderia pseudomallei and causative agent of melioidosis has been shown to regulate IL-1β and IL-18 production through NOD-like receptor NLRP3 and pyroptosis via NLRC4. Downstream signalling pathways of those receptors and other cell death mechanisms induced during B. pseudomallei infection have not been addressed so far in detail. Furthermore, the role of B. pseudomallei factors in inflammasome activation is still ill defined. In the present study we show that caspase-1 processing and pyroptosis is exclusively dependent on NLRC4, but not on NLRP3 in the early phase of macrophage infection, whereas at later time points caspase-1 activation and cell death is NLRC4- independent. In the early phase we identified an activation pathway involving caspases-9, -7 and PARP downstream of NLRC4 and caspase-1. Analyses of caspase-1/11-deficient infected macrophages revealed a strong induction of apoptosis, which is dependent on activation of apoptotic initiator and effector caspases. The early activation pathway of caspase-1 in macrophages was markedly reduced or completely abolished after infection with a B. pseudomallei flagellin FliC or a T3SS3 BsaU mutant. Studies using cells transfected with the wild-type and mutated T3SS3 effector protein BopE indicated also a role of this protein in caspase-1 processing. A T3SS3 inner rod protein BsaK mutant failed to activate caspase-1, revealed higher intracellular counts, reduced cell death and IL-1β secretion during early but not during late macrophage infection compared to the wild-type. Intranasal infection of BALB/c mice with the BsaK mutant displayed a strongly decreased mortality, lower bacterial loads in organs, and reduced levels of IL-1β, myeloperoxidase and neutrophils in bronchoalveolar lavage fluid. In conclusion, our results indicate a major role for a functional T3SS3 in early NLRC4-mediated caspase-1 activation and pyroptosis and a contribution of late caspase-1-dependent and -independent cell death mechanisms in the pathogenesis of B. pseudomallei infection.     

Tumour Necrosis Factor-alpha Production and Immune Cell Activation in Tuberculous Meningitis

The local production of tumour necrosis factor-alpha (TNFalpha) was evaluated in the cerebrospinal fluid (CSF) from ten patients with tuberculous meningitis (TBM). The degree of intrathecal immune activation was also studied by assessing the CSF levels of beta(2)-microglobulin (beta(2)-M) and adenosine deaminase activity (ADA). Results indicate that elevated CSF concentrations of TNFalpha, beta(2)-M and ADA were found in all TBM patients. Moreover, TNFalpha is produced and selectively concentrated for a long period of time, while beta(2)-M and ADA values progressively decline during the course of TBM. Our findings suggest that in TBM patients, after an early activation of immune cells, there is an enhanced and continuous production of TNFalpha at the site of infection.     

Caspase-1在炎症及程序性细胞死亡过程中的作用

Caspase家族是一类半胱氨酸天冬氨酸特异性蛋白酶,其中caspase-1是最先在哺乳动物细胞中被鉴定出来的家族成员,介导了某些特定类型细胞的凋亡。在微生物感染或细胞内危险信号存在时,caspase-1可通过与炎性体结合而发生激活,从而加工pro-IL-1β和pro-IL-18等炎症因子使其成熟并释放,在炎症反应中起着核心调控作用。此外,caspase-1还能介导一种特殊的促炎症的程序性细胞死亡(Pyroptosis)。caspase-1参与的炎症及程序性细胞死亡能有效提高机体抵抗内源和外源各种刺激的能力,达到保护宿主的目的,而caspase-1的功能异常则与多种疾病密切相关。     

Aberrant Expression of Some Circulating miRNAs in Childhood Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is a heterogeneous cancer commonly affecting children due to dysregulation of miRNA expression. In the current study, authors investigated the expression profile for miRNA-125b-1 and miRNA-203 among childhood ALL. Blood samples were collected from newly diagnosed childhood ALL and healthy control children. The expression profile for candidate miRNAs was detected using quantitative RT-PCR analysis. Statistical analysis were performed using receiver operating characteristic curve (ROC) to examine the diagnostic efficacy of the two miRNA and their levels among ALL clinicopathological factors and phenotypes. The median expression level for miRNA-125b-1 was significantly high in childhood ALL; while miRNA-203 level was significantly low in childhood ALL as compared to control ones. MiRNA-125-1 reported significant increase in T-ALL as compared to other ALL phenotypes. Median miRNA-203 level was high in T-ALL followed by pre-B-ALL although no significant difference was reported. Clinicopathological factors did not emphasize significance with either detected miRNAs. Using ROC curve the diagnostic efficacy was significant with an area under the curve 0.858 for miRNA-125b-1 (83.72, 100%) and 0.878 for miRNA-203 (97.67, 86.96%). The combination of the two key miRNAs revealed absolute sensitivity (100%). MiRNA-125b-1 and miRNA-203 can be useful molecular markers for diagnosis of ALL. Further studies with large cohort are warranted to validate these results.     

Restricted Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Pediatric B-Lineage Acute Lymphoblastic Leukemia Suggests Targetability with Therapeutic Monoclonal Antibodies

Background

Despite high cure rates for pediatric B-lineage acute lymphoblastic leukemia (B-ALL), short-term and long-term toxicities and chemoresistance are shortcomings of standard chemotherapy. Immunotherapy and chemoimmunotherapy based on monoclonal antibodies (mAbs) that target cell surface antigens with restricted expression in pediatric B-ALL may offer the potential to reduce toxicities and prevent or overcome chemoresistance. The receptor tyrosine kinase ROR1 has emerged as a candidate for mAb targeting in select B-cell malignancies.

Methodology and Principal Findings

Using flow cytometry, Western blotting, immunohistochemistry, and confocal immunofluorescence microscopy, we analyzed the cell surface expression of ROR1 across major pediatric ALL subtypes represented by 14 cell lines and 56 primary blasts at diagnosis or relapse as well as in normal adult and pediatric tissues. Cell surface ROR1 expression was found in 45% of pediatric ALL patients, all of which were B-ALL, and was not limited to any particular genotype. All cell lines and primary blasts with E2A-PBX1 translocation and a portion of patients with other high risk genotypes, such as MLL rearrangement, expressed cell surface ROR1. Importantly, cell surface ROR1 expression was found in many of the pediatric B-ALL patients with multiply relapsed and refractory disease and normal karyotype or low risk cytogenetics, such as hyperdiploidy. Notably, cell surface ROR1 was virtually absent in normal adult and pediatric tissues.

Conclusions and Significance

Collectively, this study suggests that ROR1 merits preclinical and clinical investigations as a novel target for mAb-based therapies in pediatric B-ALL. We propose cell surface expression of ROR1 detected by flow cytometry as primary inclusion criterion for pediatric B-ALL patients in future clinical trials of ROR1-targeted therapies.     

CD19 CAR-Targeted T Cells Induce Long-Term Remission and B Cell Aplasia in an Immunocompetent Mouse Model of B Cell Acute Lymphoblastic Leukemia

Although many adults with B cell acute lymphoblastic leukemia (B-ALL) are induced into remission, most will relapse, underscoring the dire need for novel therapies for this disease. We developed murine CD19-specific chimeric antigen receptors (CARs) and an immunocompetent mouse model of B-ALL that recapitulates the disease at genetic, cellular, and pathologic levels. Mouse T cells transduced with an all-murine CD3ζ/CD28-based CAR that is equivalent to the one being used in our clinical trials, eradicate B-ALL in mice and mediate long-term B cell aplasias. In this model, we find that increasing conditioning chemotherapy increases tumor eradication, B cell aplasia, and CAR-modified T cell persistence. Quantification of recipient B lineage cells allowed us to estimate an in vivo effector to endogenous target ratio for B cell aplasia maintenance. In mice exhibiting a dramatic B cell reduction we identified a small population of progenitor B cells in the bone marrow that may serve as a reservoir for long-term CAR-modified T cell stimulation. Lastly, we determine that infusion of CD8+ CAR-modified T cells alone is sufficient to maintain long-term B cell eradication. The mouse model we report here should prove valuable for investigating CAR-based and other therapies for adult B-ALL.     

The Role of Telomeres in Etoposide Induced Tumour Cell Death

Etoposide, a topoisomerase II poison is used in the treatment of a number of solidtumours. Contradictory data exist on the role of the telomere/telomerase complex inetoposide induced apoptosis. Therefore we examined the effects of etoposidetreatment in the neuroblastoma cell line SHSY5Y, with very short telomeres and theacute lymphoblastic T cell line 1301, which displays extremely long telomeres. Bothshort-term and continuous exposure to the drug was examined. Etoposide inducedwidespread DNA damage followed by DNA damage foci formation and ultimatelygrowth arrest and apoptosis in a concentration-dependent manner. However, length oftelomeres and of single stranded telomeric G rich overhangs did not changesignificantly under the treatments in any cell line. There was no significant inductionof single-strand breaks in the G- rich strand of telomeres. Telomerase activity wastransiently upregulated under low concentrations of etoposide, while highconcentrations resulted in decreased telomerase activity only after onset of apoptosis.Telomerase overexpression protected against etoposide induced apoptosis infibroblasts. The data suggest that telomeres are not major signal transducers towardsgrowth arrest or apoptosis after etoposide treatment. However, upregulation oftelomerase might be part of an attempted adaptative response, which protects cells bya mechanism that might be independent of telomere length maintenance.