Jizhong Liu

Rational engineering of L-asparaginase reveals importance of dual activity for cancer cell toxicity

Using proteins in a therapeutic context often requires engineering to modify functionality and enhance efficacy. We have previously reported that the therapeutic antileukemic protein macromolecule Escherichia coli L-asparaginase is degraded by leukemic lysosomal cysteine proteases. In the present study, we successfully engineered L-asparaginase to resist proteolytic cleavage and at the same time improve activity. We employed a novel combination of mutant sampling using a genetic algorithm in tandem with flexibility studies using molecular dynamics to investigate the impact of lid-loop and mutations on drug activity. Applying these methods, we successfully predicted the more active L-asparaginase mutants N24T and N24A. For the latter, a unique hydrogen bond network contributes to higher activity. Furthermore, interface mutations controlling secondary glutaminase activity demonstrated the importance of this enzymatic activity for drug cytotoxicity. All selected mutants were expressed, purified, and tested for activity and for their ability to form the active tetrameric form. By introducing the N24A and N24A R195S mutations to the drug L-asparaginase, we are a step closer to individualized drug design.

A dyad of lymphoblastic lysosomal cysteine proteases degrades the antileukemic drug L-asparaginase.

l-Asparaginase is a key therapeutic agent for treatment of childhood acute lymphoblastic leukemia (ALL). There is wide individual variation in pharmacokinetics, and little is known about its metabolism. The mechanisms of therapeutic failure with l-asparaginase remain speculative. Here, we now report that 2 lysosomal cysteine proteases present in lymphoblasts are able to degrade l-asparaginase. Cathepsin B (CTSB), which is produced constitutively by normal and leukemic cells, degraded asparaginase produced by Escherichia coli (ASNase) and Erwinia chrysanthemi. Asparaginyl endopeptidase (AEP), which is overexpressed predominantly in high-risk subsets of ALL, specifically degraded ASNase. AEP thereby destroys ASNase activity and may also potentiate antigen processing, leading to allergic reactions. Using AEP-mediated cleavage sequences, we modeled the effects of the protease on ASNase and created a number of recombinant ASNase products. The N24 residue on the flexible active loop was identified as the primary AEP cleavage site. Sole modification at this site rendered ASNase resistant to AEP cleavage and suggested a key role for the flexible active loop in determining ASNase activity. We therefore propose what we believe to be a novel mechanism of drug resistance to ASNase. Our results may help to identify alternative therapeutic strategies with the potential of further improving outcome in childhood ALL.

RAC2, AEP, and ICAM1 expression are associated with CNS disease in a mouse model of pre-B childhood acute lymphoblastic leukemia

We developed a murine model of CNS disease to obtain a better understanding of the pathogenesis of CNS involvement in pre-B-cell acute lymphoblastic leukemia (ALL). Semiquantitative proteomic discovery-based approaches identified unique expression of asparaginyl endopeptidase (AEP), intercellular adhesion molecule 1 (ICAM1), and ras-related C3 botulinum toxin substrate 2 (RAC2), among others, in an invasive pre-B-cell line that produced CNS leukemia in NOD-SCID mice. Targeting RAC2 significantly inhibited in vitro invasion and delayed disease onset in mice. Induced expression of RAC2 in cell lines with low/absent expression of AEP and ICAM1 did not result in an invasive phenotype or murine CNS disease. Flow cytometric analysis identified an enriched population of blast cells expressing ICAM1/lymphocyte function associated antigen-1 (LFA-1)/CD70 in the CD10(+)/CD19(+) fraction of bone marrow aspirates obtained from relapsed compared with normal controls and those with primary disease. CD10(+)/CD19(+) fractions obtained from relapsed patients also express RAC2 and give rise to CNS disease in mice. Our data suggest that combinations of processes are involved in the pathogenesis of CNS disease in pre-B-cell ALL, support a model in which CNS disease occurs as a result of external invasion, and suggest that targeting the processes of adhesion and invasion unique to pre-B cells may prevent recurrences within the CNS.

Metabolic reprogramming of bone marrow stromal cells by leukemic extracellular vesicles in acute lymphoblastic leukemia

To the editor: Cancer cells produce unique heterogeneous vesicles[1][1] capable of transferring oncogenic material[2][2],[3][3] to other cells,[4][4],[5][5] with the potential of modulating a tumor-supportive environment.[6][6][⇓][7]-[8][8] We have previously reported the presence of lipid-

Stromal cell-mediated mitochondrial redox adaptation regulates drug resistance in childhood acute lymphoblastic leukemia

Despite the high cure rates in childhood acute lymphoblastic leukemia (ALL), relapsed ALL remains a significant clinical problem. Genetic heterogeneity does not adequately explain variations in response to therapy. The chemoprotective tumor microenvironment may additionally contribute to disease recurrence. This study identifies metabolic reprogramming of leukemic cells by bone marrow stromal cells (BMSC) as a putative mechanism of drug resistance. In a BMSC-extracellular matrix culture model, BMSC produced chemoprotective soluble factors and facilitated the emergence of a reversible multidrug resistant phenotype in ALL cells. BMSC environment induced a mitochondrial calcium influx leading to increased reactive oxygen species (ROS) levels in ALL cells. In response to this oxidative stress, drug resistant cells underwent a redox adaptation process, characterized by a decrease in ROS levels and mitochondrial membrane potential with an upregulation of antioxidant production and MCL-1 expression. Similar expanded subpopulations of low ROS expressing and drug resistant cells were identified in pre-treatment bone marrow samples from ALL patients with slower response to therapy. This suggests that the bone marrow microenvironment induces a redox adaptation in ALL subclones that protects against cytotoxic stress and potentially gives rise to minimal residual disease. Targeting metabolic remodeling by inhibiting antioxidant production and antiapoptosis was able to overcome drug resistance. Thus metabolic plasticity in leukemic cell response to environmental factors contributes to chemoresistance and disease recurrence. Adjunctive strategies targeting such processes have the potential to overcome therapeutic failure in ALL.

Differential regulation of cell death pathways by the microenvironment correlates with chemoresistance and survival in leukaemia

Glucocorticoids (GCs) and topoisomerase II inhibitors are used to treat acute lymphoblastic leukaemia (ALL) as they induce death in lymphoid cells through the glucocorticoid receptor (GR) and p53 respectively. Mechanisms underlying ALL cell death and the contribution of the bone marrow microenvironment to drug response/resistance remain unclear. The role of the microenvironment and the identification of chemoresistance determinants were studied by transcriptomic analysis in ALL cells treated with Dexamethasone (Dex), and Etoposide (Etop) grown in the presence or absence of bone marrow conditioned media (CM). The necroptotic (RIPK1) and the apoptotic (caspase-8/3) markers were downregulated by CM, whereas the inhibitory effects of chemotherapy on the autophagy marker Beclin-1 (BECN1) were reduced suggesting CM exerts cytoprotective effects. GCs upregulated the RIPK1 ubiquitinating factor BIRC3 (cIAP2), in GC-sensitive (CEM-C7-14) but not in resistant (CEM-C1-15) cells. In addition, CM selectively affected GR phosphorylation in a site and cell-specific manner. GR is recruited to RIPK1, BECN1 and BIRC3 promoters in the sensitive but not in the resistant cells with phosphorylated GR forms being generally less recruited in the presence of hormone. FACS analysis and caspase-8 assays demonstrated that CM promoted a pro-survival trend. High molecular weight proteins reacting with the RIPK1 antibody were modified upon incubation with the BIRC3 inhibitor AT406 in CEM-C7-14 cells suggesting that they represent ubiquitinated forms of RIPK1. Our data suggest that there is a correlation between microenvironment-induced ALL proliferation and altered response to chemotherapy.

EVI1 expression in childhood acute lymphoblastic leukaemia is not restricted to MLL and BCR/ABL rearrangements and is influenced by age.

EVI1 expression in childhood acute lymphoblastic leukaemia is not restricted to MLL and BCR/ABL rearrangements and is influenced by age

Abstract 1506: Bone marrow microenvironment mediated redox adaptation confers drug resistance in acute lymphoblastic leukemia

Clinical and laboratory studies in childhood acute lymphoblastic leukemia (ALL) suggest that the bone marrow microenvironment protects blast cells, thus influencing disease relapse. This study was designed to identify the mechanisms by which bone marrow stromal cells could mediate chemoresistance in ALL cells. An in vitro organotypic extracellular matrix-bone marrow stromal cells (ECM-BMSC) culture system, that sustained primary cells without the addition of exogenous factors, was created. ECM-BMSC derived conditioned medium (CM) conferred broad-spectrum chemoprotection to ALL and other cancer cells. Multi drug resistant sub-clones (MDRS), derived by exposing ALL cells in CM to Mitoxantrone, retained chemoresistance even beyond 50 passages in normal medium. Chemoprotective effect was retained in the Proteinase K, RNase and heat resistant, 24 hours), pAKT activity and ROS level decreased to lower than the basal level, with upregulation of CDKN1B and MCL-1, and a decreased mitochondrial membrane potential. shRNA knockdown of CDKN1B restored normal growth rate but not chemosensitivity. MDRS that were resistant to exogenous H2O2 induced apoptosis, regained sensitivity after MCL-1 knockdown. Upregulation of ROS and downregulation of MCL-1 using PEITC lead to cell death in chemo sensitive and resistant cells. Our study identifies that the microenvironment produces soluble factors that induces a redox adaptation in ALL cells leading to chemoresistance. This can be overcome by the simultaneous inhibition of the antioxidant system (to increase ROS) and suppressing antiapoptic proteins (to eliminate the mitochondrial membrane safeguard). Tumour protection by the microenvironment is likely to be common to other cancers. Future therapeutic strategies need to consider targeting the host-tumor interactions in adjunct to conventional chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1506. doi:1538-7445.AM2012-1506