A switching mechanism in doxorubicin bioactivation can be exploited to control doxorubicin toxicity

Although doxorubicin toxicity in cancer cells is multifactorial, the enzymatic bioactivation of the drug can significantly contribute to its cytotoxicity. Previous research has identified most of the components that comprise the doxorubicin bioactivation network; however, adaptation of the network to changes in doxorubicin treatment or to patient-specific changes in network components is much less understood. To investigate the properties of the coupled reduction/oxidation reactions of the doxorubicin bioactivation network, we analyzed metabolic differences between two patient-derived acute lymphoblastic leukemia (ALL) cell lines exhibiting varied doxorubicin sensitivities. We developed computational models that accurately predicted doxorubicin bioactivation in both ALL cell lines at high and low doxorubicin concentrations. Oxygen-dependent redox cycling promoted superoxide accumulation while NADPH-dependent reductive conversion promoted semiquinone doxorubicin. This fundamental switch in control is observed between doxorubicin sensitive and insensitive ALL cells and between high and low doxorubicin concentrations. We demonstrate that pharmacological intervention strategies can be employed to either enhance or impede doxorubicin cytotoxicity in ALL cells due to the switching that occurs between oxygen-dependent superoxide generation and NADPH-dependent doxorubicin semiquinone formation.     

Identification of potential predictive markers of dexamethasone resistance in childhood acute lymphoblastic leukemia

Response to dexamethasone (DEXA), as a hallmark drug in the treatment of childhood acute lymphoblastic leukemia (ALL), is one of the pivotal prognostic factors in the prediction of outcome in ALL. Identification of predictive markers of chemoresistance is beneficial to selecting of the best therapeutic protocol with the lowest effect adverse. Hence, we aimed to find drug targets using the 2DE/MS proteomics study of a DEXA-resistant cell line (REH) as a model for poor DEXA responding patients before and after drug treatment. Using the proteomic methods, three differentially expressed proteins were detected, including voltage dependent anion channel 1 (VDAC1), sorting Nexin 3 (SNX3), and prefoldin subunit 6 (PFDN6). We observed low expression of three proteins after DEXA treatment in REH cells. We subsequently verified low expression of resulted proteins at the mRNA level using the quantitative PCR method. These proteins are promising proteins because of their important roles in drug resistance and regulation of apoptosis (VDAC1), protein trafficking (SNX3), and protein folding (PFDN6). Additionally, mRNA expression level of these proteins was assessed in 17 bone marrow samples from children with newly diagnosed ALL and 7 non-cancerous samples as controls. The results indicated that independent of the molecular subtypes of leukemia, mRNA expression of VDAC1, SNX3, and PFDN6 decreased in ALL samples compared with non-cancerous samples particularly in VDAC1 (p?<?0.001). Additionally, mRNA expression of three proteins was also declined in high-risk samples compared with standard risk cases. These results demonstrated diagnostic and prognostic value of these proteins in childhood ALL. Furthermore, investigation of protein-protein interaction using STRING database indicated that these proteins involved in the signaling pathway of NR3C1 as dexamethasone target. In conclusion, our proteomic study in DEXA resistant leukemic cells revealed VDAC1, SNX3, and PFDN6 are promising proteins that might serve as potential biomarkers of prognosis and chemotherapy in childhood ALL.     

Biochemical mechanisms of resistance to a new antineoplastic drug CRC 680578 from the nitrosourea class]

The biochemical mechanisms of resistance to CRC 680578, a new antitumour chloroethylnitrosourea alpha-amino acid derivative, were studied. Alterations in DNA, RNA and protein syntheses, SH-group content, drug efflux, activities of replicative and repair enzymes, such as ribonucleotide reductase, thymidine kinase, O6-alkylguanine-DNA-alkyltransferase and DNA polymerases alpha and beta and damages of the DNA secondary structure were investigated in sensitive and resistant to CRC 680578 leukemia L1210 cells. It was found that the total SH-group number in drug-resistant cells was increased (about 1.3-fold in comparison with sensitive cells) which seems to be due to the mechanisms of drug resistance. CHC 680578 induced less pronounced inhibition and more rapid restoration of DNA and RNA synthesis in resistant cells. No differences between the ribonucleotide reductase and thymidine kinase activities were found either in intact cells of the both strains or after drug administration. The efficiency of repair of DNA chloroethyl adducts by O6-alkylguanine-DNA-alkyltransferase in leukemia cells of various sensitivity was found to be identical. The differences in enzyme activities in intact cells of the both strains were insignificant. It was supposed that factors other than changes in the level of O6-alkylguanine-DNA-alkyltransferase in leukemia cells may be responsible for the resistance to CRC 680578. The increase in the levels of DNA polymerase alpha and, especially, of DNA polymerase beta, in sensitive (but not resistant) mouse leukemia cells 48 hours after drug administration is though to define the mechanism of resistance to the new antitumour agent CHC 680578.     

How does the novel T315L mutation of breakpoint cluster region-abelson (BCR-ABL) kinase confer resistance to ponatinib: a comparative molecular dynamics simulation study

Abstract

Acute lymphocytic leukemia (ALL) is one of the most dangerous types of leukemia, and about 40% of them is Philadelphia chromosome-positive acute lymphocytic leukemia (Ph?+?ALL). Ph?+?ALL is caused by the fusion of the breakpoint cluster region (BCR) and the Ableson (ABL) genes, named the BCR-ABL fused gene that codes for an autonomously active tyrosine kinase. Tyrosine kinase inhibitors (TKIs) are among the first-line therapeutic agents for the treatment of Ph?+?ALL. Drug resistance are the major obstacle, limiting their clinical utility. The latest third-generation TKIs, ponatinib, can tackle most abnormal BCR-ABL kinases, including the T315I mutant that is resistant to first- and second-generations TKIs such as imatinib. However, drug resistance still emerges with the novel T315L mutation and the underlying mechanisms remain elusive. Here, using molecular dynamics (MD) simulations, we explored into the detailed interactions between ponatinib and BCR-ABL in the wild-type (WT), T315I, and T315L systems. The simulations revealed the significant conformational changes of ponatinib in its binding site due to the T315L mutation and the underlying structural mechanisms. Binding free energy analysis unveiled that the affinity of ponatinib to BCR-ABL decreased upon T315L mutation, which resulted in its unfavorable binding and drug resistance. Key residues responsible for the unfavored unbinding were also identified. This study elucidates the detailed mechanisms for the resistance of ponatinib in Ph?+?ALL triggered by the T315L mutation and will provide insights for future drug development and optimization.     

Metabolic adaptations of Leishmania donovani in relation to differentiation,drug resistance,and drug pressure

Antimonial (sodium stibogluconate, SSG) resistance and differentiation have been shown to be closely linked in Leishmania donovani, with SSG‐resistant strains showing an increased capacity to generate infectious (metacyclic) forms. This is the first untargeted LC‐MS metabolomics study which integrated both phenomena in one experimental design and provided insights into metabolic differences between three clinical L. donovani strains with a similar genetic background but different SSG‐susceptibilities. We performed this analysis at different stages during promastigote growth and in the absence or presence of drug pressure. When comparing SSG‐resistant and SSG‐sensitive strains, a number of metabolic changes appeared to be constitutively present in all growth stages, pointing towards a clear link with SSG‐resistance, whereas most metabolic changes were only detected in the stationary stage. These changes reflect the close intertwinement between SSG‐resistance and an increased metacyclogenesis in resistant parasites. The metabolic changes suggest that SSG‐resistant parasites have (i) an increased capacity for protection against oxidative stress; (ii) a higher fluidity of the plasma membrane; and (iii) a metabolic survival kit to better endure infection. These changes were even more pronounced in a resistant strain kept under Sb^III drug pressure.     

Proteomic analysis reveals a novel role for the actin cytoskeleton in vincristine resistant childhood leukemia--an in vivo study

Intrinsic or acquired resistance to vincristine (VCR), an antimicrotubule agent used in the treatment of childhood acute lymphoblastic leukemia (ALL), is a major clinical problem. Using a clinically relevant NOD/SCID mouse xenograft model of ALL, we established that alterations in the actin and tubulin cytoskeleton are involved in in vivo VCR resistance. Altered protein expression between VCR-sensitive ALL xenografts, and xenografts with intrinsic or acquired VCR resistance, was identified using 2-D DIGE coupled with MS. Of the 19 proteins displaying altered expression, 11 are associated with the actin cytoskeleton. Altered expression of the actin- and/or tubulin-binding proteins gelsolin, moesin, ezrin, tropomyosin, CAP-G, HSP27, HSP70, TCP-1, and stathmin were associated with in vivo VCR resistance. The actin-regulating protein gelsolin was increased in both acquired and resistant leukemia as confirmed by immunoblotting and gene expression. The major cytoskeletal protein, gamma-actin, was down-regulated in the VCR-resistant leukemia xenografts; in contrast, there was no significant change in beta-actin expression. This study provides the first evidence for a role of the actin cytoskeleton in intrinsic and acquired in vivo antimicrotubule drug resistance in childhood leukemia and highlights the power of 2-D DIGE for the discovery of resistance markers, pharmacoproteomics, and signaling pathways in cancer.     

Che‐1 is targeted by c‐Myc to sustain proliferation in pre‐B‐cell acute lymphoblastic leukemia

Despite progress in treating B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL), disease recurrence remains the main cause of treatment failure. New strategies to improve therapeutic outcomes are needed, particularly in high‐risk relapsed patients. Che‐1/AATF (Che‐1) is an RNA polymerase II‐binding protein involved in proliferation and tumor survival, but its role in hematological malignancies has not been clarified. Here, we show that Che‐1 is overexpressed in pediatric BCP‐ALL during disease onset and at relapse, and that its depletion inhibits the proliferation of BCP‐ALL cells. Furthermore, we report that c‐Myc regulates Che‐1 expression by direct binding to its promoter and describe a strict correlation between Che‐1 expression and c‐Myc expression. RNA‐seq analyses upon Che‐1 or c‐Myc depletion reveal a strong overlap of the respective controlled pathways. Genomewide ChIP‐seq experiments suggest that Che‐1 acts as a downstream effector of c‐Myc. These results identify the pivotal role of Che‐1 in the control of BCP‐ALL proliferation and present the protein as a possible therapeutic target in children with relapsed BCP‐ALL.     

BCL2 inhibitor ABT-199 and JNK inhibitor SP600125 exhibit synergistic cytotoxicity against imatinib-resistant Ph+ ALL cells

Imatinib (IMT), a specific tyrosine kinase inhibitor (TKI), has drastically changed the treatment strategy for Ph+ ALL (Philadelphia chromosome-positive acute lymphoblastic leukemia). However, TKI resistance remains a serious problem for patient prognosis. Here, a Ph+ ALL cell line NphA2 and the IMT-resistant subline NphA2/STIR were analyzed to identify a potential novel treatment strategy. We also examined other Ph+ ALL cells, MR87 and its IMT-resistant subline, MR87/STIR. IMT induced apoptosis of NphA2 and MR87 but had no effect on resistant sublines. Increased phosphorylated ERK and BCL2, but not BCL-XL, were observed in NphA2/STIR compared with NphA2. NphA2/STIR but not NphA2 was moderately sensitive to U0126, an ERK inhibitor. Interestingly, SP600125, a JNK inhibitor, was potent in cell growth inhibition and apoptosis induction of both parental and IMT-resistant NphA2 and MR87 cells. Moreover, NphA2 and MR87 and their IMT-resistant sublines were sensitive to ABT-199, a specific BCL2 inhibitor. The combination of SP600125 and ABT-199 synergistically suppressed both parental and IMT-resistant cells, including one with T315I mutation, suggesting that Ph+ ALL exhibits high sensitivity to ABT-199 and SP600125 regardless of TKI resistance. This combination might be a possible therapeutic strategy for Ph+ ALL in the future.     

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.     

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.     

Metabolic rewiring in drug resistant cells exhibit higher OXPHOS and fatty acids as preferred major source to cellular energetics

Alteration in metabolic repertoire is associated with resistance phenotype. Although a common phenotype, not much efforts have been undertaken to design effective strategies to target the metabolic drift in cancerous cells with drug resistant properties. Here, we identified that drug resistant AML cell line HL-60/MX2 did not follow classical Warburg effect, instead these cells exhibited drastically low levels of aerobic glycolysis. Biochemical analysis confirmed reduced glucose consumption and lactic acid production by resistant population with no differences in glutamine consumption. Raman spectroscopy revealed increased lipid and cytochrome content in resistant cells which were also visualized as lipid droplets by Raman mapping, electron microscopy and lipid specific staining. Gene set enrichment analysis data from sensitive and resistant cell lines revealed significant enrichment of lipid metabolic pathways in HL-60/MX2 cells. Further, HL-60/MX2 possessed higher mitochondrial activity and increased OXPHOS suggesting the role of fatty acid metabolism as energy source which was confirmed by increased rate of fatty acid oxidation. Accordingly, OXPHOS inhibitor increased sensitivity of resistant cells to chemotherapeutic drug and fatty acid oxidation inhibitor Etomoxir reduced colony formation ability of resistant cells demonstrating the requirement of fatty acid metabolism and dependency on OXPHOS by resistant leukemic cells for survival and tumorigenicity.     

The Transcription Factor Wilms Tumor 1 Confers Resistance in Myeloid Leukemia Cells against the Proapoptotic Therapeutic Agent TRAIL (Tumor Necrosis Factor α-related Apoptosis-inducing Ligand) by Regulating the Antiapoptotic Protein Bcl-xL

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is considered a promising cancer therapeutic agent due to its ability to induce apoptosis in a variety of cancer cells, while sparing normal cells. However, many human tumors including acute myeloid leukemia (AML) are partially or completely resistant to monotherapy with TRAIL, limiting its therapeutic utility. Therefore, identification of factors that contribute to TRAIL resistance may facilitate future development of more effective TRAIL-based cancer therapies. Here, we report a previously unknown role for WT1 in mediating TRAIL resistance in leukemia. Knockdown of WT1 with shRNA rendered TRAIL-resistant myeloid leukemia cells sensitive to TRAIL-induced cell death, and re-expression of shRNA-resistant WT1 restored TRAIL resistance. Notably, TRAIL-mediated apoptosis in WT1-silenced cells was largely due to down-regulation of the antiapoptotic protein Bcl-xL. Moreover, WT1 expression strongly correlated with overexpression of Bcl-xL in AML cell lines and blasts from AML patients. Furthermore, we found that WT1 transactivates Bcl-xL by directly binding to its promoter. We previously showed that WT1 is a novel client protein of heat shock protein 90 (Hsp90). Consistent with this, pharmacological inhibition of Hsp90 resulted in reduced WT1 and Bcl-xL expression leading to increased sensitivity of leukemia cells to TRAIL-mediated apoptosis. Collectively, our results suggest that WT1-dependent Bcl-xL overexpression contributes to TRAIL resistance in myeloid leukemias.     

Antileukemic effects of topoisomerase I inhibitors mediated by de-SUMOylase SENP1

SUMO-specific proteases (SENPs) play pivotal roles in maintaining the balance of SUMOylation/de-SUMOylation and in SUMO recycling. Deregulation of SENPs leads to cellular dysfunction and corresponding diseases. As a key member of the SENP family, SENP1 is highly correlated with various cancers. However, the potential role of SENP1 in leukemia, especially in acute lymphoblastic leukemia (ALL), is not clear. This study shows that ALL cells knocking down SENP1 display compromised growth rather than significant alterations in chemosensitivity, although ALL relapse samples have a relatively higher expression of SENP1 than the paired diagnosis samples. Camptothecin derivatives 7-ethylcamptothecin (7E-CPT, a monomer compound) and topotecan (TPT, an approved clinical drug) induce specific SENP1 reduction and severe apoptosis of ALL cells, showing strong anticancer effects against ALL. Conversely, SENP1 could attenuate this inhibitory effect by targeting DNA topoisomerase I (TOP1) for de-SUMOylation, indicating that specific reduction in SENP1 induced by 7E-CPT and/or topotecan inhibits the proliferation of ALL cells.     

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.     

miRNA-155 and miRNA-181a as prognostic biomarkers for pediatric acute lymphoblastic leukemia

Mortality rates of acute lymphoblastic leukemia (ALL) have improved over the past 20 years; however, a significant portion of deaths stems from the lack of prognostic biomarkers, which can direct therapy and overcome drug resistance. microRNA-155a (miRNA-155a) and miRNA-181a are two single-stranded miRNAs involved in the pathogenesis of many types of leukemia and lymphoma and is linked to drug resistance. We investigated their expression levels in 55 patients, 45 diagnosed with ALL and 10 as a control group. We found that miRNA-155a and miRNA-181a were significantly upregulated in the ALL group with both being linked to high levels of minimal residual disease and poor prognosis. miRNA-155a cutoff value was significant in discriminating between high- and low-risk ALL patients as well as between ALL patients and healthy controls, miRNA-181a cutoff value, however, was not significant. Both markers levels were significantly downregulated after therapy. We conclude that miR-155 is correlated with poor prognosis in ALL, whereas we couldn’t link miRNA-181a to the prognosis in ALL. Moreover, the marked decrease in their expression after therapy could reflect their impact on disease outcome.     

Multi-Agent Chemotherapy Overcomes Glucocorticoid Resistance Conferred by a BIM Deletion Polymorphism in Pediatric Acute Lymphoblastic Leukemia

A broad range of anti-cancer agents, including glucocorticoids (GCs) and tyrosine kinase inhibitors (TKIs), kill cells by upregulating the pro-apoptotic BCL2 family member, BIM. A common germline deletion in the BIM gene was recently shown to favor the production of non-apoptotic BIM isoforms, and to predict inferior responses in TKI-treated chronic myeloid leukemia (CML) and EGFR-driven lung cancer patients. Given that both in vitro and in vivo GC resistance are predictive of adverse outcomes in acute lymphoblastic leukemia (ALL), we hypothesized that this polymorphism would mediate GC resistance, and serve as a biomarker of poor response in ALL. Accordingly, we used zinc finger nucleases to generate ALL cell lines with the BIM deletion, and confirmed the ability of the deletion to mediate GC resistance in vitro. In contrast to CML and lung cancer, the BIM deletion did not predict for poorer clinical outcome in a retrospective analysis of 411 pediatric ALL patients who were uniformly treated with GCs and chemotherapy. Underlying the lack of prognostic significance, we found that the chemotherapy agents used in our cohort (vincristine, L-asparaginase, and methotrexate) were each able to induce ALL cell death in a BIM-independent fashion, and resensitize BIM deletion-containing cells to GCs. Together, our work demonstrates how effective therapy can overcome intrinsic resistance in ALL patients, and suggests the potential of using combinations of drugs that work via divergent mechanisms of cell killing to surmount BIM deletion-mediated drug resistance in other cancers.     

HDAC inhibitors induce apoptosis in glucocorticoid-resistant acute lymphatic leukemia cells despite a switch from the extrinsic to the intrinsic death pathway

Inhibitors of histone deacetylases (HDACi's) are promising novel tools for cancer therapy. We have compared the growth inhibitory and apoptogenic potential of the pan-HDACi SAHA and the sub-class I selective HDAC inhibitor MS275, as well as valproic acid (VPA) on glucocorticoid sensitive and resistant B (B-ALL) and T (T-ALL) cell acute lymphoblastic leukemia cells and patients blasts. In contrast, to our previous results with U937 acute myeloid leukemia (AML) cells which showed a similar activity of MS275 and SAHA in growth inhibition and apoptosis induction, both B and T-ALL cells were much more efficiently killed by SAHA and VPA than by MS275. The same relative potency was observed with some patient ALL blasts treated ex vivo. SAHA displayed similar efficacy on glucocorticoid-sensitive and insensitive ALL cells but did not synergize with dexamethasone. In studying mediators of apoptosis we found that the TRAIL receptor DR5 is constitutively expressed in glucocorticoid-sensitive CEM-C7 cells which are also TRAIL sensitive. In contrast, glucocorticoid-insensitive CEM-C1 cells do not express DR5 and are insensitive to TRAIL. However, SAHA induces, in addition to p21(WAF1/CIP1) also re-expression of DR5. Importantly, SAHA-induced apoptosis of CEM-C7 cells operates through initiator caspase 10, while it induces apoptosis of CEM-C1 cells through the intrinsic, as well as through caspase-independent death pathways. Our data suggest that the generation of resistance to glucocorticoids has dramatically altered death signaling in these cells and that SAHA overcomes these restrictions by inducing alternative death pathways.