Acute Lymphoblastic Leukemia Drug Research Articles

Bacterial Expression of TMTP1-Fused L-Asparaginase for Targeting Leukemia and Metastatic Tumor Cells

L-asparaginase is recognized as a first-line anticancer drug for acute lymphoblastic leukemia (ALL); however, low-substrate specificity and exhibiting glutaminase activity cause various off-target toxicities on normal cells. In the following study, we functionalized wild-type asparaginase with the TMTP1 targeting peptide which specifically targets a variety of hematological and metastatic cancer cells. The peptide sequence was genetically added to the N-terminal end of the asparaginase using the restriction endonuclease-free cloning method. Wild-type and engineered asparaginases were expressed in E. coli and purified by Nickel affinity chromatography column. The in vitro activity of both types of enzymes was evaluated by Nessler’s method. The sequencing results showed that the TMTP1 sequence was added in the correct frame to the asparaginase. Wild-type and TMTP1-fused asparaginases were produced in a soluble state with the specific activity of 172 U/mg and 153 U/mg, respectively. The evidence from this study suggests that TMTP1-fused asparaginase could preserve its solubility and activity compared to the wild-type species and can be proposed for future research in anticancer therapies.

L-Asparaginase regulates mTORC1 activity via a TSC2-dependent pathway in pancreatic beta cells

Eif2ak4, a susceptibility gene for type 2 diabetes, encodes GCN2, a molecule activated by amino acid deficiency. Mutations or deletions in GCN2 in pancreatic β-cells increase mTORC1 activity by decreasing Sestrin2 expression in a TSC2-independent manner. In this study, we searched for molecules downstream of GCN2 that suppress mTORC1 activity in a TSC2-dependent manner. To do so, we used a pull-down assay to identify molecules that competitively inhibit the binding of the T1462 phosphorylation site of TSC2 to 14-3-3. l-asparaginase was identified. Although l-asparaginase is frequently used as an anticancer drug for acute lymphoblastic leukemia, little is known about endogenous l-asparaginase. l-Asparaginase, which is expressed downstream of GCN2, was found to bind 14-3-3 and thereby to inhibit its binding to the T1462 phosphorylation site of TSC2 and contribute to TSC2 activation and mTORC1 inactivation upon TSC2 dephosphorylation. Further investigation of the regulation of mTORC1 activity in pancreatic β-cells by l-asparaginase should help to elucidate the mechanism of diabetes and insulin secretion failure during anticancer drug use.

The Correlation Between Children’s Acute Lymphoblastic Leukemia Drug Resistance System Induced by Metabolomics-Based 6-Mercaptopurine and Hypoxanthine-Guanine Phosphoribosyl Transferase 1 Protein

This study aimed to explore 6-mercaptopurine (MP)-induced children’s acute lymphoblastic leukemia (ALL) drug resistance system and leukemia hypoxanthine-guanine phosphoribosyl transferase 1 (HPRT1) protein. Based on metabonomics, drug resistance of 6MP-Reh cell line was established by increasing concentration administration method, and the degree of drug resistance of 6MP-Reh was verified by apoptosis test, western blotting (WB) test, and drug sensitivity test. The changes of tissue inhibitor of matrix metalloproteinase (TIMP) and thioguanosine monophosphate (TGMP) in drug-resistant cells were detected through liquid chromatograph (LC)/mass spectrometer (MS). The 6MP-Reh-wt cell line was established by lentivirus infection, so as to verify the correlation between HPRT1 and drug resistance mechanism. The results showed that the inhibition concentration (IC50) value, cell vitality (CV), apoptosis rate, and 6-MP content of 6MP-Reh were higher hugely than those of Reh (P < 0.05). The contents of HPRT1, TIMP, and TGMP in 6MP-Reh cells were lower sharply than the contents of Reh cells (P < 0.001). The IC50 value of 6MP-Reh-wt was also lower steeply than the value of 6MP-Reh (P < 0.001), and the concentrations of TIMP and TGMP increased obviously (P < 0.05). Therefore, it indicated that the mutation of HPRT1 in drugresistant cell lines could lead to a decrease in their viability and cause leukemia cells to develop resistance to 6-MP. In addition, HPRT1 gene could improve their resistance to 6-MP.

Analysis of acute lymphoblastic leukemia drug sensitivity by changes in impedance via stromal cell adherence

The bone marrow is a frequent location of primary relapse after conventional cytotoxic drug treatment of human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Because stromal cells have a major role in promoting chemotherapy resistance, they should be included to more realistically model in vitro drug treatment. Here we validated a novel application of the xCELLigence system as a continuous co-culture to assess long-term effects of drug treatment on BCP-ALL cells. We found that bone marrow OP9 stromal cells adhere to the electrodes but are progressively displaced by dividing patient-derived BCP-ALL cells, resulting in reduction of impedance over time. Death of BCP-ALL cells due to drug treatment results in re-adherence of the stromal cells to the electrodes, increasing impedance. Importantly, vincristine inhibited proliferation of sensitive BCP-ALL cells in a dose-dependent manner, correlating with increased impedance. This system was able to discriminate sensitivity of two relapsed Philadelphia chromosome (Ph) positive ALLs to four different targeted kinase inhibitors. Moreover, differences in sensitivity of two CRLF2-drivenBCP-ALL cell lines to ruxolitinib were also seen. These results show that impedance can be used as a novel approach to monitor drug treatment and sensitivity of primary BCP-ALL cells in the presence of protective microenvironmental cells.

Comprehensive analysis of dose intensity of acute lymphoblastic leukemia chemotherapy

Chemotherapy dosages are often compromised, but most reports lack data on dosages that are actually delivered. In two consecutive acute lymphoblastic leukemia trials that differed in their asparaginase formulation, native E. coli L-asparaginase in St. Jude Total 15 (T15, n=365) and pegaspargase in Total 16 (T16, n=524), we tallied the dose intensities for all drugs on the low-risk or standard-risk arms, analyzing 504,039 dosing records. The median dose intensity for each drug ranged from 61-100%. Dose intensities for several drugs were more than 10% higher on T15 than on T16: cyclophosphamide (P<0.0001 for the standard- risk arm), cytarabine (P<0.0001 for the standard-risk arm), and mercaptopurine (P<0.0001 for the low-risk arm and P<0.0001 for the standardrisk arm). We attributed the lower dosages on T16 to the higher asparaginase dosages on T16 than on T15 (P<0.0001 for both the low-risk and standard-risk arms), with higher dose-intensity for mercaptopurine in those with anti-asparaginase antibodies than in those without (P=5.62x10- 3 for T15 standard risk and P=1.43x10-4 for T16 standard risk). Neutrophil count did not differ between protocols for low-risk patients (P=0.18) and was actually lower for standard-risk patients on T16 than on T15 (P<0.0001) despite lower dosages of most drugs on T16. Patients with low asparaginase dose intensity had higher methotrexate dose intensity with no impact on prognosis. The only dose intensity measure predicting a higher risk of relapse on both studies was higher mercaptopurine dose intensity, but this did not reach statistical significance (P=0.03 T15; P=0.07 T16). In these intensive multiagent trials, higher dosages of asparaginase compromised the dosing of other drugs for acute lymphoblastic leukemia, particularly mercaptopurine, but lower chemotherapy dose intensity was not associated with relapse.

Development and Validation of a Hydrophilic Interaction Liquid Chromatography Tandem Mass Spectrometry Method for the Determination of Asparagine in Human Serum.

L-Asparagine (ASN) is the catalyze substrate of L-asparaginase (ASNase), which is an important drug for acute lymphoblastic leukemia (ALL) patients. The ASN level is found to be closely associated with the effectiveness of ASNase treatment. In this study, a hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) method was developed for the determination of ASN in the human serum using a stable isotope-labeled internal standard (ASN-D3). Serum samples were prepared by a one-step precipitation procedure using methanol and separated by an Agilent HILIC Plus column with the mobile phase of methanol-water (95 : 5, v/v, containing 5 mM ammonium formate and 0.1% formic acid), at a constant flow rate of 0.3 mL/min. Mass spectrometric analysis was conducted using multiple-reaction monitoring in the positive electrospray ionization mode. Serum ASN concentrations were determined over a linear calibration curve range of 2–200 μM, with acceptable accuracies and precisions. The validated HILIC-MS/MS method was successfully applied to the quantification of ASN levels in the serum from patients with ALL. Collectively, the research may shed new light on an alternative rapid, simple, and convenient quantitative method for determination of serum ASN in ALL patients treated with ASNase.

Influence of Cyclophosphamide on L-Asparaginase-Induced Allergy in Animal Model

BACKGROUND: L-Asparaginase (L-ASP) is one of the essential drugs for acute lymphoblastic leukemia and lymphoma. Since L-ASP proteins used clinically are derived from bacteria, they often cause allergies, including anaphylaxis. However, the characteristics of L-ASP-induced hypersensitivity are not well documented so far. Also, there is neither suitable treatment for hypersensitivity to L-ASP nor adequate experimental model of L-ASP allergy. In addition, some anti-cancer drugs, used concomitantly with L-ASP, are known to affect lymphocyte activities, although the role of such drugs on L-ASP hypersensitivity is not known. Previously, we have established mouse model of L-ASP allergy and found that a neutralizing antibody (Ab) of IgE is effective to inhibit L-ASP-induced allergic reaction. Aims: In order to establish in vitro model of L-ASP allergy, we used RBL-2H3 mast cells and evoked degranulation of the cells sensitized with L-ASP-allergic mouse serum using L-ASP as an antigen. Furthermore, we examined the effect of cyclophosphamide (CY), which reportedly impairs the functions of regulatory T (Treg) cells, on L-ASP allergy both in vivo and in vitro. Methods: Male BALB/c mice (7 week old) were sensitized with L-ASP (100 units) with Al(OH)3 gel (1 mg) on days 1 and 15. The right ears of the mice were locally sensitized on days 18, 21, 24 by i.d. injection of L-ASP (10 units). Antigen challenge was carried out on day 27 by i.d. injection (10 units). CY (75, 150, 300 mg/kg) was i.p. administrated at day -1 and day 13. Anti-IgE treatment (BD Bioscience, clone R35-92; 100 μg, i.p.) was carried out on the day before L-ASP challenge. The serum was collected on day 27. Total IgE level in the serum was measured by ELISA kit (Yamasa). RBL-2H3 cells were sensitized by the serum and stimulated by L-ASP to determine β-hexosaminidase (β-Hex) release in vitro. Results : L-ASP-sensitization induced allergic ear edema and an increase in serum IgE level in mice, both of which are augmented by an administration of CY at 150 mg/kg. L-ASP-induced allergic ear edema was inhibited by a pretreatment with anti-IgE Ab. L-ASP-induced CY-enhanced ear edema was also inhibited by anti-IgE Ab. On the other hand, at 300 mg/kg of CY, L-ASP-induced ear edema was much lower than 150 mg/kg group, though IgE concentration in the serum was as high as CY 150 mg/kg group. When RBL-2H3 cells were sensitized by anti-L-ASP serum, L-ASP challenge induced β-Hex release. Anti-L-ASP serum of CY 150 mg/kg-treated mice induced higher β-Hex release than normal anti-L-ASP sera in vitro, though that of CY 300 mg/kg-treated mice did not induce β-Hex release. After removing IgG from the serum of CY 300 mg/kg-treated mice using protein G Mag Sepharose (GE Healthcare), β-Hex release became higher than normally sensitized mice. Anti-IgE Ab treatment both ex vivo and in vitro inhibited L-ASP-induced β-Hex release from the RBL-2H3 cells sensitized by anti-L-ASP serum of CY-treated mice. Conclusions: In this study, we showed that L-ASP sensitization induced IgE production in vivo and this serum could bring about β-Hex release from RBL-2H3 cells in vitro; both allergic reactions were inhibited by an anti-IgE antibody which binds to Fc region of IgE molecule. From these results, we propose that non-anaphylactogenic neutralizing antibody of IgE, such as omalizumab, can be a candidate drug for the treatment of L-ASP hypersensitivity. Also in this study, CY induced biphasic effects on L-ASP allergy in mice, enhancing it at low dosage and attenuating it at high dosage. Since CY is reportedly impair the functions of Treg cells, CY-induced suppression of Treg may enhance Th2 responses so as to augment L-ASP hypersensitivity. Further investigations for the detection and avoidance of L-ASP hypersensitivity are under the way. Disclosures No relevant conflicts of interest to declare.

Transcription factor E2F3a regulates CASP8AP2 transcription and enhances sensitivity to chemotherapeutic drugs in acute lymphoblastic leukemia

BackgroundLow expression of E2F3a and caspase 8 associated protein 2 (CASP8AP2) are associated with poor prognosis of childhood acute lymphoblastic leukemia (ALL).MethodsDual-luciferase reporter assay and wild type as well as four mutated types of reporter plasmids were used to demonstrate the activation of E2F3a on CASP8AP2 transcription. The direct binding of E2F3a with the promoter of CASP8AP2 was shown by Chromatin Immunoprecipitation (ChIP). Cell proliferation activity and cell cycle were determined by MTS and flow cytometry in leukemic cells after treating with common chemotherapeutic drugs vincristine and daunorubicin.ResultsIn this study, we found that up-regulation of E2F3a in leukemic cells led to increased fraction of cells in S and G2/M phase, accelerated proliferation, and enhanced sensitivity to vincristine and daunorubicin. ChIP and luciferase assay indicated that E2F3a could directly bind to two fragments in the wild type of CASP8AP2 promotor (− 206 to − 69 and − 677 to − 507), and activate its transcription activity which was reduced in mutated promotors. The effect of E2F3a on chemotherapeutic sensitivity of leukemic cells could be reversed by down-regulating CASP8AP2.ConclusionsE2F3a could promote transcription and expression of CASP8AP2. The effect of E2F3a on chemotherapeutic sensitivity of ALL cells was implemented by regulating CASP8AP2 expression to a great extent.

Regulation of p53 and survivin by prodigiosin compound derived from Serratia marcescens contribute to caspase-3-dependent apoptosis in acute lymphoblastic leukemia cells.

Tumor suppressor p53 and proto-oncogene survivin are challenging targets for anticancer drugs in acute lymphoblastic leukemia (ALL) which are associated with chemoresistance. Yet, no p53 and survivin-modulating drug with low toxicity and high efficacy has been approved for clinical application in ALL. Consequently, the search for novel compounds which target p53 or survivin is needed to further advance ALL treatment. Prodigiosin, a secondary metabolite of Serratia marcescens induces apoptosis in cancer cells with no toxicity on normal cells. However, the possible potential of prodigiosin as p53- and survivin-modulating agent in ALL cells has not been investigated. Wt-p53 Molt-4 cells were treated with 100 to 600 nM prodigiosin, after which, viability, cell proliferation rates, survivin and p53 protein levels, caspase-3 activation, and apoptosis were evaluated. After 24-, 48-, and 72-h treatments with 100 to 600 nM prodigiosin, cell proliferation rates were measured to be 93.7-77.3%, 75.5-58.3%, and 55-23.3%, respectively. Treatment for 48 hours with 100 to 600 nM prodigiosin resulted in 41-19% decrease in survivin protein levels followed by 450-950% increases in caspase-3 activation levels. Prodigiosin induced remarkably p53 accumulation and increased p53/survivin and caspase-3/survivin ratios by 6.1 to 11.3 and 10.3 to 47.5-fold at 100 to 600 nM, respectively. Survivin protein levels were inversely proportional to p53 accumulation levels. Low survivin protein levels combined with high levels of p53 accumulation were correlated to higher apoptotic rates. P53 and survivin as molecular targets of prodigiosin contribute to caspase-3-dependent apoptosis in ALL cells and this compound represents an attractive p53- and survivin-modulating agent in ALL.

Toxicity profile of anticancer drugs in acute lymphoblastic leukemia and protective role of vitamin E on these toxicities in albino rats

&lt;p class="abstract"&gt;&lt;strong&gt;Background:&lt;/strong&gt; Objectives:&lt;strong&gt; &lt;/strong&gt;(1) To study the spectrum of toxicity due to chemotherapy as per MCP841 protocol during the acute phase of treatment of patients with Acute Lymphoblastic Leukemia (ALL).&lt;strong&gt; &lt;/strong&gt;(2) To study the same toxicities in albino rats and the protective role of vitamin E on these toxicities.&lt;strong&gt;&lt;/strong&gt;&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Methods:&lt;/strong&gt; This was a prospective clinical study for one year, in the department of medical oncology and paediatric oncology which was augmented by experimental animal study for 5 months using albino rats. For clinical study, patients diagnosed as ALL taking treatment as per MCP841 protocol were taken. Patients were followed up every day to detect the development of any type of adverse reactions or toxicity symptoms. Laboratory tests done during the course of chemotherapy were reviewed for any abnormality. Animal study involved 18 albino rats; rats were divided into 3 Groups; Group 1 - control (n=6), Group 2 - antileukaemic treated rats (n=6), Group 3 - antileukaemic drugs and vitamin-E treated rats (n=6). &lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Results:&lt;/strong&gt; In the clinical study major toxicities observed were haematological, metabolic, gastrointestinal, general infection, neurotoxicity, pancreatitis, pneumonitis and jaundice. Neurosensory toxicity presented as numbness in the extremities and hyperalgesia and myalgia. Histopathological examination of the internal organs of albino rats studied showed&lt;strong&gt; &lt;/strong&gt;protection of vitamin E for the gastric toxicity, pancreatitis, cardiac toxicity, neuro toxicity and hepatic toxicity whereas there was no reduction in splenic and renal toxicities. In the case of haematological toxicity, protection was only minimal.&lt;/p&gt;&lt;p class="abstract"&gt;&lt;strong&gt;Conclusions:&lt;/strong&gt; The animal study revealed the protective role of vitamin E on cytostatic drug induced toxicities. &lt;/p&gt;&lt;strong&gt;Keywords: &lt;/strong&gt;Acute lymphoblastic leukaemia (ALL), Antileukaemic drugs, Vitamin E (Vit. E), MCP841 protocol

Novel and Emerging Drugs for Acute Lymphoblastic Leukemia

Over the past several decades the important progress has been made in the management of acute lymphoblastic leukemia (ALL), especially among children. However, in adult patients reported cure rates seldom exceeded 40%, despite the use of hematopoietic stem-cell transplantation in many cases. Conventional chemotherapy is toxic and ineffective. Therefore, new treatment options and risk-adapted therapies are needed to improve the outcome of ALL patients. This review is focused on new systemic treatment modalities, such as nucleoside analogues, purine synthetase inhibitors, monoclonal antibodies, tyrosine kinase inhibitors and other agents targeting molecular pathways that are aimed to benefit patients and improve the outcome of their treatment.

Methylated chrysin reduced cell proliferation, but antagonized cytotoxicity of other anticancer drugs in acute lymphoblastic leukemia

The efficacy of 5,7-dimethoxyflavone (DMF), a methylated analog of chrysin, as a therapeutic agent to treat acute lymphoblastic leukemia (ALL) was investigated. Using a panel of ALL cell lines, the IC50 (half-maximal inhibitory concentration) of DMF varied between 2.8 and 7.0 μg/ml. DMF induced G0/G1 cell cycle arrest, concomitant with a decreased expression of phosphorylated retinoblastoma-associated protein 1. DMF increased the rate of apoptosis, although it was apparent only after a long period of exposure (96 h). The accumulation of oxidative stress was not involved in the growth-inhibitory effects of DMF. As DMF reduced the intracellular levels of glutathione, the combination effects of DMF with other anticancer drugs were evaluated using the improved Isobologram and the combination index method. In the simultaneous drug combination assay, DMF antagonized the cytotoxicity of 4-hydroperoxy-cyclophosphamide, cytarabine, vincristine, and L-asparaginase in all tested ALL cells. This study demonstrated that DMF, a methylated flavone, was an effective chemotherapy agent that could inhibit cell cycle arrest and induce apoptosis in ALL cell lines. However, combination therapy with DMF and other anticancer drugs is not recommended.

Population Analysis of Pharmacogenetic Polymorphisms Related to Acute Lymphoblastic Leukemia Drug Treatment

This study aimed to evaluate in the Brazilian population, the genotypes and population frequencies of pharmacogenetic polymorphisms involved in the response to drugs used in treatment of acute lymphoblastic leukemia (ALL), and to compare the data with data from the HapMap populations. There was significant differentiation between most population pairs, but few associations between genetic ancestry and SNPs in the Brazilian population were observed. AMOVA analysis comparing the Brazilian population to all other populations retrieved from HapMap pointed to a genetic proximity with the European population. These associations point to preclusion of the use of genetic ancestry as a proxy for predicting drug response. In this way, any study aiming to correlate genotype with drug response in the Brazilian population should be based on pharmacogenetic SNP genotypes.

A Genome-Wide Approach Identifies Variations in the Aspartate Metabolism Pathway That Are Associated with Asparaginase Sensitivity

Asparaginase is an important drug for acute lymphoblastic leukemia (ALL). The basis for interindividual differences in asparaginase sensitivity remains unclear. To comprehensively identify genetic variants important in asparaginase sensitivity, we employed a genome-wide association approach using the HapMap lymphoblastoid cell lines from 87 individuals of European ancestry (CEU) and diagnostic ALL blasts from 42 newly diagnosed, genomically-determined white patients. In vitro sensitivity was based on IC50 values measured following 48 hour exposures to native E. coli asparaginase (0.003–100 IU/ml) in CEU cell lines and 96 hour exposures (0.003–10 IU/ml) in patient samples using the methylthiazol tetrazolium assay. For CEU cell lines, single nucleotide polymorphism (SNP) genotypes were downloaded from the International HapMap database (www.hapmap.org) and gene expression data (Affymetrix GeneChip Human Exon 1.0 ST Array) were downloaded from http://www.ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE7761. For patients with ALL, we used the 500K SNP arrays to interrogate germline DNA and Affymetrix U133A GeneChip Array to assess gene expression in ALL blasts. We tested whether 2,390,203 SNP genotypes were associated with asparaginase IC50 using a linear mixed effect model in CEU cell lines, setting a p value threshold of p < 0.001 for individual SNPs and p < 0.05 at the gene level. This approach yielded 329 SNPs representing 94 genes. Combining these germline SNPs with those representing genes whose expression was also associated with IC50 at the p < 0.05 level (1,706 genes), there were 6 SNPs representing 5 genes, two of which (rs8135371 and rs17001863, both in the ADSL gene) contributed to asparaginase sensitivity (p = 6.9 × 10−4 and 9.1 × 10− 4, respectively) through their effects on ADSL gene expression. The top ranked KEGG pathway overrepresented by the 94 top-ranked genes (329 SNPs) was that of aspartate metabolism, which may be directly linked to the mechanism of action of asparaginase. The two most highly ranked genes (ADSL and DARS) in this pathway encompassed 7 SNPs (rs8135371, rs17001863, rs3768998, rs2278683, rs11893318, rs2322725, and rs7587285), all with p < .001. Using multiple linear regression analysis, 32% of the variability in asparaginase IC50 among the CEU cell lines could be accounted for by these 7 SNPs (p = 5.9 × 10−7). To examine the overall contribution of the aspartate metabolism pathway to asparaginase IC50, we compared all SNPs (935 in cell lines, 717 in patients) representing the aspartate pathway with those SNPs representing other pathways, using a random forest model. We found that the SNP genotypes in the aspartate pathway explained significantly more variation in asparaginase IC50 in cell lines (11.4%, p = 6.9 × 10−4) and in ALL patient samples (11.2%, p = 0.02) than other pathways. The expression of ADSL differed among ALL subtypes, with more sensitive subtypes (hyperdiploid and TEL-AML1 ALL) having lower ADSL expression than more resistant subtypes (T-ALL) (p = 1.1 × 10−5 and 2.9 × 10−9, respectively). Genome-wide interrogation of CEU cell lines and primary ALL blasts revealed that inherited and acquired genomic interindividual variation in a plausible candidate pathway contribute to asparaginase sensitivity.