Effect of cytosine arabinoside metabolizing enzyme expression on drug toxicity in acute myeloid leukemia

No abstarc

Role of NF-E2 related factor 2 (Nrf2) on chemotherapy resistance in acute myeloid leukemia (AML) and the effect of pharmacological inhibition of Nrf2

<div><p>Cytarabine (Ara-C) and Daunorubicin (Dnr) forms the backbone of acute myeloid leukemia (AML) therapy. Drug resistance and toxic side effects pose a major threat to treatment success and hence alternate less toxic therapies are warranted. NF-E2 related factor-2 (Nrf2), a master regulator of antioxidant response is implicated in chemoresistance in solid tumors. However, little is known about the role of Nrf2 in AML chemoresistance and the effect of pharmacological inhibitor brusatol in modulating this resistance. Primary AML samples with high <i>ex-vivo</i> IC50 to Ara-C, ATO, Dnr had significantly high <i>NRF2</i> RNA expression. Gene-specific knockdown of <i>NRF2</i> improved sensitivity to these drugs in resistant AML cell lines by decreasing the expression of downstream antioxidant targets of Nrf2 by compromising the cell’s ability to scavenge the ROS. Treatment with brusatol, a pharmacological inhibitor of Nrf2, improved sensitivity to Ara-C, ATO, and Dnr and reduced colony formation capacity. AML cell lines stably overexpressing <i>NRF2</i> showed increased resistance to ATO, Dnr and Ara-C and increased expression of downstream targets. This study demonstrates that Nrf2 could be an ideal druggable target in AML, more so to the drugs that function through ROS, suggesting the possibility of using Nrf2 inhibitors in combination with chemotherapeutic agents to modulate drug resistance in AML.</p></div

ATP-binding casette transporter expression in acute myeloid leukemia: association with <i>in vitro</i> cytotoxicity and prognostic markers

Introduction: Drug resistance and relapse are considered to be the major reasons for treatment failure in acute myeloid leukemia (AML). There is limited data on the role of ABC transporter expression on in vitro sensitivity to cytarabine (Ara-C) and daunorubicin (Dnr) in primary AML cells. Patients &#38; methods: RNA expression levels of 12 ABC transporters were analyzed by real-time quantitative PCR in 233 de novo adult acute myeloid leukemia patients. Based on cytarabine or Dnr IC₅₀, the samples were categorized as sensitive, intermediate and resistant. Role of candidate ABC transporter RNA expression on in vitro cytotoxicity, treatment outcome post therapy as well as the influence of various prognostic markers on ABC transporter expression were analyzed. Results: Expression of ABCC3 and ABCB6 were significantly higher in Dnr-resistant samples when compared with Dnr-sensitive samples. Increased ABCC1 expression was associated with poor disease-free survival in this cohort of patients. Conclusion: This comprehensive analysis suggests ABCC1, ABCC3, ABCB6 and ABCA5 as probable targets which can be modulated for improving chemotherapeutic responses

Carbonyl reductase 1 expression influences daunorubicin metabolism in acute myeloid leukemia

Purpose: The present study aimed to investigate the role of expression of daunorubicin-metabolizing enzymes carbonyl reductase 1 and 3 (CBR1 and CBR3) on the in vitro cytotoxicity of daunorubicin in primary acute myeloid leukemia (AML) cells and the effect of genetic variants in CBR1 and CBR3 on the plasma pharmacokinetics of daunorubicin and daunorubicinol (DOL) in AML patients. Methods: RNA expression of CBR1 and CBR3, intracellular daunorubicin and DOL levels, and in vitro cytotoxicity of daunorubicin were measured in bone marrow mononuclear cells of 104 adult AML patients. Plasma pharmacokinetics of daunorubicin and DOL was measured in 24 patients receiving daunorubicin-based induction chemotherapy for AML. Results: Increased expression of CBR1 significantly reduced the in vitro cytotoxicity of daunorubicin and also positively correlated with intracellular DOL levels. Polymorphisms in CBR1 and CBR3 did not show any association with intracellular daunorubicin or DOL levels, but there was a trend towards significant increase in plasma daunorubicin systemic exposure in patients with a variant genotype for CBR1 polymorphism rs25678. Conclusions: This pilot study suggests that CBR1 RNA expression may be helpful in identifying AML patients at risk of developing resistance or toxicity to daunorubicin due to increased formation of DOL. Further confirmation of these findings in a larger sample pool would be required to determine the applicability of these results. Inhibition of CBR1 can be an option to improve the efficacy and prevent toxicity related to the treatment. Influence of daunorubicin and DOL plasma levels on clinical outcome, if any, remains to be evaluated

Cytidine deaminase genetic variants influence RNA expression and cytarabine cytotoxicity in acute myeloid leukemia

Aim: Cytidine deaminase (CDA) irreversibly deaminates cytarabine (Ara-C), a key component of acute myeloid leukemia (AML) induction and consolidation therapy. CDA overexpression results in Ara-C resistance, while decreased expression is associated with toxicity. We evaluated factors influencing variation in CDA mRNA expression in adult AML patients and normal controls, and how they contributed to Ara-C cytotoxicity in AML cells. Materials and methods: CDA mRNA expression in 100 de novo AML patients and 36 normal controls were determined using quantitative reverse-transcriptase PCR. Genetic variants in the CDA gene were screened by direct sequencing. IC50 of Ara-C was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: CDA RNA expression as well as Ara-C IC50 showed wide variation in AML samples and normal controls. Fourteen sequence variants were identified, three of which (-33delC, intron 2 TCAT repeat and the 3´untranslated region 816delC variants) showed significant association with RNA expression and the nonsynonymous coding variant 79A&#62;C was associated with Ara-C cytotoxicity. Conclusion: CDA genetic variants explain the variation in RNA expression and may be candidates for individualizing Ara-C therapy

<i>NRF2</i> overexpression in AML cell lines increases IC50 of ATO & Dnr by up-regulating downstream target expression.

<p>Two AML cell lines (HL-60 and MOLM-13) with relatively low expression of <i>NRF2</i> were made to stably over express <i>NRF2</i>. <b>(A)</b> Overexpression was further confirmed by quantitative real-time PCR (i) and western blot (ii) for Nrf2 <b>(B)</b> Nrf2 downstream target genes (<i>GCLC</i>, <i>GCLM</i>, <i>NQO1</i>, <i>and HMOX-1</i>) were analyzed by real time quantitative PCR. <b>(C)</b> <i>In-vitro</i> cytotoxicity of overexpressed cells to Dnr and ATO was compared with control cells. Overexpression increases resistance to Dnr and ATO.</p

Primary AML cells and cell lines resistant to cytarabine, daunorubicin and arsenic trioxide show increased Nrf2 expression.

<p><b>(A)</b> AML samples categorised based on median IC50 values to Ara-C (6μM), Dnr (0.4μM) and ATO (2.42μM) were analysed for the expression of <i>NRF2</i> by quantitative real time PCR. Y axis denotes relative expression (2^-dCT) of <i>NRF2</i> normalised to <i>GAPDH</i>. <b>(B)</b> <i>NRF2</i> levels in AML cell lines resistant (THP1, U937) or sensitive (HL-60, MOLM-13) to Ara-C, Dnr and ATO were analysed and represented as fold change (2^-ddCT) normalised to Molm13 (with low <i>NRF2</i> RNA)} (n = 5). *Statistical significance were calculated based on Kruskal–Wallis test and p<0.05 is indicated. <b>(C)</b> Flow cytometric analysis of total Nrf2 expression in AML cell lines (n = 4). <b>(D)</b> Immunofluorescence analysis of nuclear Nrf2 expression in AML cell lines {4′,6-diamidino-2-phenylindole (blue) and Nrf2 antibody (red) stained images overlay} (n = 2). <b>(E)</b> Immunoblotting of total Nrf2 in AML cell lines (β-actin was used as loading control) (n = 3). <b>(F)</b> Nrf2 downstream target expression {(<i>HO-1</i>, <i>NQO1</i>, <i>GCLC</i> and <i>GCLM</i>) normalized to <i>GAPDH</i> and expressed relative to that of MOLM-13 cell line (n = 4)}.*Statistical significance were calculated based on Kruskal–Wallis test and p<0.05 is indicated.</p

Brusatol treatment brings down Nrf2 expression and reduces colony forming capacity of THP1 cells.

<p>THP1 cells were treated with 1μM of brusatol reconstituted in DMSO (final concentration of 0.01%) for 6hrs. The expression of Nrf2 after treatment with brusatol was assessed by <b>(A)</b> flow cytometry (n = 2) (i), western blot with β—actin as the loading control (n = 3) (ii) and subcellular expression (iii). <b>(B)</b> THP1 cells were treated with 100nM of Brusatol for 6h and downstream targets of Nrf2 <i>GCLC</i>, <i>GCLM</i>, <i>HO-1</i>, <i>and NQO1</i> was evaluated by quantitative real time PCR (n = 4). RNA expression of all target genes was normalized to <i>GAPDH</i>. *Statistical significance were calculated based on unpaired t test and p<0.05 is indicated. <b>(C)</b> Apoptosis upon incubation with two different concentrations 100nM and 1000nM of Brusatol was assessed by Annexin V-7AAD.</p

Pharmacological inhibition of Nrf2 with brusatol brings down the IC50 of Ara-C, Dnr & ATO in resistant AML cell lines.

<p>THP1 cells were incubated with Nrf2 inhibitor Brusatol 100nM for 6hrs, followed by increasing concentration of <b>(A)</b> Ara-C (0.1–80μM), <b>(B)</b> Dnr (0.0025–1μM) and <b>(C)</b> ATO (0.1–6μM) for 48hrs. <i>In-vitro</i> cytotoxicity was measured by MTT assay (n = 9). <b>(D)</b> IC50 of THP1 to Dnr and ATO was compared with untreated control cells. <b>(E)</b> THP1 cells were incubate with 100nM of Brusatol followed by treatment with 5μM of Ara-C, 0.25μM of Dnr or 6μM of ATO for 24hrs. Control and treated (5*10³) cells were seeded in methyl cellulose medium (n = 2) and enumerated on day 14. Light microscopic images of colonies were taken on day 14 (10X magnification) (i). Control or treated (5*10³) cells seeded in methyl cellulose medium were stained with methylene blue (ii) and colonies were enumerated (n = 2)(iii). Brusatol alone minimally reduces the colony forming capacity of THP1 cells and considerably reduces in combination with Ara-C, Dnr and ATO.</p

RNA expression of genes involved in cytarabine metabolism and transport predicts cytarabine response in acute myeloid leukemia

Background: Variation in terms of outcome and toxic side effects of treatment exists among acute myeloid leukemia (AML) patients on chemotherapy with cytarabine (Ara-C) and daunorubicin (Dnr). Candidate Ara-C metabolizing gene expression in primary AML cells is proposed to account for this variation. Methods:Ex vivo Ara-C sensitivity was determined in primary AML samples using MTT assay. mRNA expression of candidate Ara-C metabolizing genes were evaluated by RQPCR analysis. Global gene expression profiling was carried out for identifying differentially expressed genes between exvivo Ara-C sensitive and resistant samples. Results: Wide interindividual variations in ex vivo Ara-C cytotoxicity were observed among samples from patients with AML and were stratified into sensitive, intermediately sensitive and resistant, based on IC50 values obtained by MTT assay. RNA expression of deoxycytidine kinase (DCK), human equilibrative nucleoside transporter-1 (ENT1) and ribonucleotide reductase M1 (RRM1) were significantly higher and cytidine deaminase (CDA) was significantly lower in ex vivo Ara-C sensitive samples. Higher DCK and RRM1 expression in AML patient's blast correlated with better DFS. Ara-C resistance index (RI), a mathematically derived quotient was proposed based on candidate gene expression pattern. Ara-C ex vivo sensitive samples were found to have significantly lower RI compared with resistant as well as samples from patients presenting with relapse. Patients with low RI supposedly highly sensitive to Ara-C were found to have higher incidence of induction death (p = 0.002; RR: 4.35 [95% CI: 1.69–11.22]). Global gene expression profiling undertaken to find out additional contributors of Ara-C resistance identified many apoptosis as well as metabolic pathway genes to be differentially expressed between Ara-C resistant and sensitive samples. Conclusion: This study highlights the importance of evaluating expression of candidate Ara-C metabolizing genes in predicting ex vivo drug response as well as treatment outcome. RI could be a predictor of ex vivo Ara-C response irrespective of cytogenetic and molecular risk groups and a potential biomarker for AML treatment outcome and toxicity