Mass spectrometric quantification of asparagine synthetase in circulating leukemia cells from acute lymphoblastic leukemia patients

The appearance of asparaginase-resistant acute lymphoblastic leukemia (ALL) in transformed cell lines has been correlated with increased expression of asparagine synthetase (ASNS). Recent measurements using mRNA-based assays have raised doubts, however, as to the importance of ASNS protein in the cellular mechanisms that confer drug resistance upon the leukemic cells. Studies aimed at determining the concentration of ASNS protein in human leukemias are therefore needed to resolve this issue. A mass spectrometry (MS)-based procedure is presented for the direct quantification of ASNS protein concentration in complex sample mixtures. This assay is able to distinguish samples from transformed cell lines that express ASNS over a wide dynamic range of concentration. Importantly, this method directly detects ASNS protein, the functional entity that may be synthesizing sufficient asparagine to render leukemia cells resistant to asparaginase-treatment. We also report the successful use of this MS method, which has lower limits of detection and quantification of 30 and 100 attomoles, respectively, for the first direct measurements of ASNS protein concentrations in four patient blast samples.     

A critical electrostatic interaction mediates inhibitor recognition by human asparagine synthetase

The first sulfoximine-based inhibitor of human asparagine synthetase (ASNS) with nanomolar potency has been shown to suppress proliferation of asparaginase-resistant MOLT-4 cells in the presence of l-asparaginase. This validates literature hypotheses concerning the viability of human ASNS as a target for new drugs against acute lymphoblastic leukemia and ovarian cancer. Developing structure–function relationships for this class of human ASNS inhibitors has proven difficult, however, primarily because of the absence of rapid synthetic procedures for constructing highly functionalized sulfoximines. We now report conditions for the efficient preparation of these compounds by coupling sulfoxides and sulfamides in the presence of a rhodium catalyst. Access to this methodology has permitted the construction of two new adenylated sulfoximines, which were expected to exhibit similar binding affinity and better bioavailability than the original human ASNS inhibitor. Steady-state kinetic characterization of these compounds, however, has revealed the importance of a localized negative charge on the inhibitor that mimics that of the phosphate group in a key acyl-adenylate reaction intermediate. These experiments place an important constraint on the design of sulfoximine libraries for screening experiments to obtain ASNS inhibitors with increased potency and bioavailability.     

Preclinical testing of the Akt inhibitor triciribine in T‐cell acute lymphoblastic leukemia

Over the past 20 years, survival rates of T‐cell acute lymphoblastic leukemia (T‐ALL) patients have improved, mainly because of advances in polychemotherapy protocols. Despite these improvements, we still need novel and less toxic treatment strategies targeting aberrantly activated signaling networks which increase proliferation, survival, and drug resistance of T‐ALL cells. One such network is represented by the phosphatidylinositol 3‐kinase (PI3K)/Akt axis. PI3K inhibitors have displayed some promising effects in preclinical models of T‐ALL. Here, we have analyzed the therapeutic potential of the Akt inhibitor, triciribine, in T‐ALL cell lines. Triciribine caused cell cycle arrest and caspase‐dependent apoptosis. Western blots demonstrated a dose‐dependent dephosphorylation of Akt1/Akt2, and of mammalian target of rapamycin complex 1 downstream targets in response to triciribine. Triciribine induced autophagy, which could be interpreted as a defensive mechanism, because an autophagy inhibitor (chloroquine) increased triciribine‐induced apoptosis. Triciribine synergized with vincristine, a chemotherapeutic drug employed for treating T‐ALL patients, and targeted the side population of T‐ALL cell lines, which might correspond to leukemia initiating cells. Our findings indicate that Akt inhibition, either alone or in combination with chemotherapeutic drugs, may serve as an efficient treatment towards T‐ALL cells requiring upregulation of this signaling pathway for their proliferation and survival. J. Cell. Physiol. 226: 822–831, 2011. © 2010 Wiley‐Liss, Inc.     

Distribution of the cytoskeletal protein,Nestin, in acute leukemia

Abstract

Nestin is a neuroepithelial stem cell marker that is expressed in some types of tumor cells. Recent reports suggest that Nestin may be closely related to malignant cell proliferation and migration. Acute leukemia (AL) is characterized by a lack of differentiation, which results in uncontrolled proliferation in the bone marrow and accumulation of immature cells. The expression and function of Nestin in AL is unclear. We investigated Nestin immunohistochemical patterns of 87 patients that included 47 cases of acute myeloid leukemia (AML) and 40 cases of acute lymphoblastic leukemia (ALL), and 20 patients in complete remission (CR) from AML or ALL. We also investigated the clinico-pathological features of 87 cases of AL and their CR and overall survival (OS). Nestin was expressed in leukemic blasts and mature granulocytic cells in most cases (39/47) of AML. Conversely, Nestin was expressed in mature granulocytic cells in fewer cases (6/40) of ALL, but not in blasts. Nestin expression appeared in leukemic blasts of AML, but not ALL. Nestin expression in AML blast cells was not associated with CR or OS. We provide evidence that Nestin is expressed in AL and might be a useful immunohistochemical marker for identifying AML and ALL.     

Small Molecule ErbB Inhibitors Decrease Proliferative Signaling and Promote Apoptosis in Philadelphia Chromosome–Positive Acute Lymphoblastic Leukemia

The presence of the Philadelphia chromosome in patients with acute lymphoblastic leukemia (Ph⁺ALL) is a negative prognostic indicator. Tyrosine kinase inhibitors (TKI) that target BCR/ABL, such as imatinib, have improved treatment of Ph⁺ALL and are generally incorporated into induction regimens. This approach has improved clinical responses, but molecular remissions are seen in less than 50% of patients leaving few treatment options in the event of relapse. Thus, identification of additional targets for therapeutic intervention has potential to improve outcomes for Ph+ALL. The human epidermal growth factor receptor 2 (ErbB2) is expressed in ∼30% of B-ALLs, and numerous small molecule inhibitors are available to prevent its activation. We analyzed a cohort of 129 ALL patient samples using reverse phase protein array (RPPA) with ErbB2 and phospho-ErbB2 antibodies and found that activity of ErbB2 was elevated in 56% of Ph⁺ALL as compared to just 4.8% of Ph⁻ALL. In two human Ph+ALL cell lines, inhibition of ErbB kinase activity with canertinib resulted in a dose-dependent decrease in the phosphorylation of an ErbB kinase signaling target p70S6-kinase T389 (by 60% in Z119 and 39% in Z181 cells at 3 µM). Downstream, phosphorylation of S6-kinase was also diminished in both cell lines in a dose-dependent manner (by 91% in both cell lines at 3 µM). Canertinib treatment increased expression of the pro-apoptotic protein Bim by as much as 144% in Z119 cells and 49% in Z181 cells, and further produced caspase-3 activation and consequent apoptotic cell death. Both canertinib and the FDA-approved ErbB1/2-directed TKI lapatinib abrogated proliferation and increased sensitivity to BCR/ABL-directed TKIs at clinically relevant doses. Our results suggest that ErbB signaling is an additional molecular target in Ph⁺ALL and encourage the development of clinical strategies combining ErbB and BCR/ABL kinase inhibitors for this subset of ALL patients.     

Genome-Wide Analysis of Small RNA and Novel MicroRNA Discovery in Human Acute Lymphoblastic Leukemia Based on Extensive Sequencing Approach

Background

MicroRNAs (miRNAs) have been proved to play an important role in various cellular processes and function as tumor suppressors or oncogenes in cancers including leukemia. The identification of a large number of novel miRNAs and other small regulatory RNAs will provide valuable insights into the roles they play in tumorgenesis.

Methodology/Principal Findings

To gain further understanding of the role of miRNAs relevant to acute lymphoblastic leukemia (ALL), we employed the sequencing-by-synthesis (SBS) strategy to sequence small RNA libraries prepared from ALL patients and normal donors. In total we identified 159 novel miRNAs and 116 novel miRNA*s from both libraries. Among the 159 novel miRNAs, 42 were identified with high stringency in our data set. Furthermore, we demonstrated the different expression patterns of 20 newly identified and several known miRNAs between ALL patients and normal donors, suggesting these miRNAs may be associated with ALL and could constitute an ALL-specific miRNA signature. Interestingly, GO “biological process” classifications revealed that a set of significantly abnormally expressed miRNAs are associated with disease relapse, which implies that these dysregulated miRNAs might promote the progression of ALL by regulating genes involved in the pathway of the disease development.

Conclusion/Significance

The study presents a comprehensive picture of the expression of small RNAs in human acute lymphoblastic leukemia and highlights novel and known miRNAs differentially expressed between ALL patients and normal donors. To our knowledge, this is the first study to look at genome-wide known and novel miRNA expression patterns in in human acute lymphoblastic leukemia. Our data revealed that these deregulated miRNAs may be associated with ALL or the onset of relapse.     

Acute lymphoblastic leukemia-derived extracellular vesicles affect quiescence of hematopoietic stem and progenitor cells

Patient-derived xenografted (PDX) models were generated through the transplantation of primary acute lymphoblastic leukemia (ALL) cells into immunodeficient NSG mice. We observed that ALL cells from mouse bone marrow (BM) produced extracellular vesicles (EVs) with specific expression of inducible heat shock protein HSP70, which is commonly activated in cancer cells. Taking advantage of this specific expression, we designed a strategy to generate fluorescent HSP70-labeled ALL EVs and monitor the impact of these EVs on endogenous murine BM cells ex vivo and in vivo. We discovered that hematopoietic stem and progenitor cells (HSPC) were mainly targeted by ALL EVs, affecting their quiescence and maintenance in the murine BM environment. Investigations revealed that ALL EVs were enriched in cholesterol and other metabolites that contribute to promote the mitochondrial function in targeted HSPC. Furthermore, using CD34⁺ cells isolated from cord blood, we confirmed that ALL EVs can modify quiescence of human HSPC. In conclusion, we have discovered a new oncogenic mechanism illustrating how EVs produced by proliferative ALL cells can target and compromise a healthy hematopoiesis system during leukemia development.Subject terms: Acute lymphocytic leukaemia, Cancer models, Cancer metabolism     

Philadelphia chromosome-positive acute lymphoblastic leukemia- current concepts and future perspectives

Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) is diagnosed rarely in children, but constitutes the most frequent cytogenetic abnormality in adults with ALL. In contrast to chronic myeloid leukemia (CML), patients with Ph-positive ALL usually demonstrate expression of a truncated version of the BCR-ABL protein called p190bcr-abl. Irrespective of age and breakpoint location, Ph-positive ALL carries a poor prognosis. Although remission rates are identical to those of Ph-negative ALL, relapse is almost universal and long-term survival remains rare. Given the poor outcome with current chemotherapy consolidation programs, stem cell transplantation is usually recommended for these patients in first remission or as soon as feasible. Even with transplantation the impact on outcome is limited and new therapeutic concepts are urgently needed. One of the most promising developments in recent years has been the introduction of the tyrosine kinase inhibitors such as STI571. An overview of current treatment modalities in Ph-positive ALL will be provided and the rationale for new therapies will be discussed.     

RAD001 (Everolimus) induces autophagy in acute lymphoblastic leukemia

The elimination of tumor cells by apoptosis is the main mechanism of action of chemotherapeutic drugs used in current treatment protocols of acute lymphoblastic leukemia (ALL). Despite the rapid cytoreduction achieved, serious acute and late complications are frequent, and resistance to chemotherapy develops. During the past decade, new strategies to kill cancer cells by non-apoptotic mechanisms have flourished and many mediators of alternate cell death pathways have been identified. In the present study we have evaluated the efficacy of an mTOR inhibitor, RAD001 (Everolimus), to induce autophagy in an in vivo model of childhood ALL. In particular we found that RAD001 increased Beclin 1 expression, the conversion of the soluble form of microtubule-associated protein 1 light chain 3 (LC3) to the autophagic vesicle-associated form LC3-II and the occurence of lysosomes/autophagosomes. Focal degradation of cytoplasmic areas sequestered by autophagic structures was demonstrated by electron microscopy. This effect was associated with massive reduction of leukemic mass and a strong survival advantage for mice treated with RAD001. The discovery of alternative pathways involved in cell death execution and the role that it plays in leukemia suggest mTOR inhibitors should be included in future chemotherapy protocols of ALL.     

Epigenetic regulation of human gamma-glutamyl hydrolase activity in acute lymphoblastic leukemia cells

Gamma-glutamyl hydrolase (GGH) catalyzes degradation of the active polyglutamates of natural folates and the antifolate methotrexate (MTX). We found that GGH activity is directly related to GGH messenger RNA expression in acute lymphoblastic leukemia (ALL) cells of patients with a wild-type germline GGH genotype. We identified two CpG islands (CpG1 and CpG2) in the region extending from the GGH promoter through the first exon and into intron 1 and showed that methylation of both CpG islands in the GGH promoter (seen in leukemia cells from approximately 15% of patients with nonhyperdiploid B-lineage ALL) is associated with significantly reduced GGH mRNA expression and catalytic activity and with significantly higher accumulation of MTX polyglutamates (MTXPG(4-7)) in ALL cells. Furthermore, methylation of CpG1 was leukemia-cell specific and had a pronounced effect on GGH expression, whereas methylation of CpG2 was common in leukemia cells and normal leukocytes but did not significantly alter GGH expression. These findings indicate that GGH activity in human leukemia cells is regulated by epigenetic changes, in addition to previously recognized genetic polymorphisms and karyotypic abnormalities, which collectively determine interindividual differences in GGH activity and influence MTXPG accumulation in leukemia cells.     

Serum amino acid levels in leukemic mice after L-asparaginase treatment

A rapid, reproducible HPLC method based on dansyl chloride derivatization has been developed for the determination of L-asparagine, L-aspartate, L-glutamine, and L-glutamate in mouse and human serum samples. This improved procedure has been designed for automation with an autoinjector system. Studies with mice bearing the sensitive and the asparaginase-resistant L5178Y leukemia show that this analytical method can be employed to monitor the effect of L-asparaginase on serum levels of these four amino acids. The method can be used to monitor serum amino acid levels in patients undergoing therapy with L-asparaginase.     

Disparate in vivo efficacy of FTY720 in xenograft models of Philadelphia positive and negative B-lineage acute lymphoblastic leukemia

Most patients with acute lymphoblastic leukemia (ALL) respond well to standard chemotherapy-based treatments. However a significant proportion of patients, particularly adult patients, relapse with the majority dying of leukemia. FTY720 is an immunosuppressive drug that was recently approved for the treatment of multiple sclerosis and is currently under pre-clinical investigation as a therapy for a number of hematological malignancies. Using human ALL xenografts in NOD/SCIDγc(-/-) mice, we show for the first time that three Ph(+) human ALL xenografts responded to FTY720 with an 80 ± 12% (p = 0.048) reduction in overall disease when treatment was commenced early. In contrast, treatment of mice with FTY720 did not result in reduced leukemia compared to controls using four separate human Ph(-) ALL xenografts. Although FTY720 reactivated PP2A in vitro, this reactivation was not required for death of Ph(-) ALL cells. The plasma levels of FTY720 achieved in the mice were in the high nanomolar range. However, the response seen in the Ph(+) ALL xenografts when treatment was initiated early implies that in vivo efficacy may be obtained with substantially lower drug concentrations than those required in vitro. Our data suggest that while FTY720 may have potential as a treatment for Ph(+) ALL it will not be a useful agent for the treatment of Ph(-) B-ALL.     

抑制GSK-3β活性促进NF-κB诱导的儿童急性淋巴细胞白血病细胞凋亡

核转录因子-κB(NF-κB)是维持急性淋巴细胞白血病(ALL)细胞生存的关键因子.近年来发现,糖原合成酶激酶-3β(GSK-3β)可以正性调控NF-κB的活性.本研究通过抑制GSK-3β活性初步探讨ALL细胞中GSK-3β在NF-κB诱导细胞凋亡中的作用机制.收集ALL患儿骨髓单个核细胞,采用免疫荧光细胞化学方法检测到ALL细胞核内GSK-3β有明显聚集.体外培养ALL细胞后经GSK-3β抑制剂氯化锂(LiCl)和SB216763处理,采用Western印迹和EMSA检测发现,ALL细胞核内GSK-3β表达下降,而NF-κBP65蛋白无明显变化,但是其活性明显降低.同时RT-PCR结果显示,NF-κB下游抗凋亡基因存活素(survivin)的表达随之下降,AnnexinV-PE/7-AAD双染流式细胞仪检测结果证实,ALL细胞凋亡明显增加(P0.01).该结果表明,抑制GSK-3β活性可以下调NF-κB的转录活性,并通过下调抗凋亡基因存活素的表达而促进ALL细胞的凋亡.