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In low-resource settings, Cryptosporidium spp. is a common cause of diarrheal disease in children under the age of 3 years. In addition to diarrhea, these children also experience subclinical episodes that have been shown to affect growth and cognitive function. In this study, we screened polymorphisms in the promoter and exon1 regions of the mannose binding lectin 2 (MBL2) gene, as well as single nucleotide polymorphisms (SNPs) described in toll-like receptors (TLR) TLR1, TLR2, TLR4, and TLR9 and TIR domain-containing adaptor protein (TIRAP) genes among children with cryptosporidial diarrhea (cases) and children who only experienced asymptomatic (subclinical) cryptosporidiosis (controls). Among the polymorphisms screened, the variant allele B at codon 54 (rs1800450) of the MBL2 gene was associated with susceptibility to cryptosporidial diarrhea (odds ratio [OR] = 2.2, 95% confidence interval [CI] 1.1–4.5). When plasma MBL levels were compared, 72% of cases were found to be deficient compared with 32% among controls (OR = 5.09). Among TLR polymorphisms screened, multivariate analysis showed that heterozygous genotypes of TLR4 896A/G (rs4986790, OR = 0.33, 95% CI: 0.11–0.98) and TIRAP 539 C/T (rs8177374, OR = 0.19, 95% CI: 0.06–0.64) SNPs were associated with protection from cryptosporidial diarrhea. Although not statistically significant, these findings suggest that polymorphisms of MBL2 and TLR genes influence susceptibility to symptomatic cryptosporidial diarrhea even in settings with high exposure levels. Further studies to validate these findings in a larger cohort and to understand the role of these polymorphisms in mediating innate and adaptive immune responses to cryptosporidial infection are necessary.

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. Original submitted 22 December 2014; Revision submitted 9 April 2015

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 & 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.

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 & 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>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. Original submitted 8 July 2011; Revision submitted 10 October 2011

Although 70-80% of Acute Myeloid Leukemia (AML) patients achieve complete remission with the induction therapy consisting of a combination of Cytarabine (Ara-C) and Daunorubicin (Dnr), the overall survival is dismal with disease relapse, drug resistance and toxicities as the major causes of treatment failure. Single agent arsenic trioxide (ATO) used in acute promyelocytic leukemia (APL or AML-M3) is associated with relatively less toxicities which suggest the possibility of extending its use to elderly non M3-AML patients. But ATO trials in non-M3 AML were not encouraging enough to devise ATO as a better treatment option. NF-E2 related factor 2 (NRF2) has been recognised as one of the key molecules associated with resistance towards chemotherapeutic agents in cancer. Leukemic cells owing to their genomic instability and altered metabolism are known to maintain high reactive oxygen species (ROS) levels. NRF2 as a redox dependent transcription factor drives the expression of several antioxidant genes like NAD(P)H quinone oxidoreductase1 (NQO1), heme oxygenase1 (HMOX1), glutamate-cysteine ligase (GCL) and glutathione-S-transferases (GSTs) which coordinate to scavenge ROS. Although the role of NRF2 in resistance to cytarabine and daunorubicin has been reported previously, its role in ATO resistance in non M3-AML has not been addressed before. Also, the role of pharmacological inhibition of NRF2 in modulating resistance to ATO and other conventional therapeutic agents in AML has not been tested. To investigate whether NRF2 expression is associated with in-vitro sensitivity to chemotherapeutic drugs, NRF2 RNA expression in primary AML samples grouped according to their median Ara-C, Dnr and ATO IC50 (median IC50 6uM for Ara-C, 0.4µM for Dnr and 2.42µM for ATO) were compared. Primary AML samples with Ara-C, Dnr or ATO IC50 below median had significantly low NRF2 RNA expression compared to those above median (p=0.07, 0.004 and 0.01, respectively) (Fig1-A). Comparison of NRF2 expression in a subset of samples that were above median IC50 to both ATO and Dnr with those below median also showed similar trend. Flow cytometric evaluation of NRF2 expression in Ara-C, Dnr and ATO resistant AML cell lines (THP1 and U937) showed higher intracellular NRF2 levels (MFI=37.18 and 46.56) compared to sensitive cell lines (HL60 and MOLM13) (MFI=6.25 and 28.9). Treatment of resistant AML cell lines expressing high levels of NRF2 (THP1 and U937) with pharmacological inhibitor of NRF2 {Brusatol} followed by ATO (0.1-6uM), Ara-C (0.1uM-80uM) and Dnr (25nM-2000nM) improved their sensitivity to these drugs (Fig1-B) The effect of Brusatol in inhibiting NRF2 protein levels and its role in inducing apoptosis were also demonstrated by flow cytometry (Fig1-C). To further confirm the role of NRF2 in drug resistance, AML cell lines (MOLM13 and HL60) stably over expressing NRF2 was established. Overexpression was done using lentiviral expression system followed by blasticidin selection, further confirmed by immunoblot and quantitative real time PCR. The overexpressed cell lines showed increased resistance to ATO/ Dnr and Ara-C (Fig1-D) and up-regulation of NRF2 downstream targets compared to un-transduced cells (Fig1-E). Our results suggest that NRF2 plays a pivotal role in drug resistance and hence could be an ideal druggable target in AML, more so to the drugs that functions through ROS. Our data as well growing evidence from other malignancies suggest the possibility of using NRF2 inhibitors in combination with chemotherapeutic agents to combat drug resistance. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Daunorubicin (Dnr) has been the choice of anthracycline in the induction chemotherapy of Acute Myeloid Leukemia (AML) along with Cytarabine for the past 4 decades. Drug resistance and relapse of the disease has been the major concern in the success of AML therapy. Several factors, including pharmacokinetics (PK) of Dnr could have impact on treatment outcome as pharmacokinetic effects might limit the amount of drug that reaches the tumor. In this study, we analyzed the factors influencing plasma PK of Daunorubicin and its major metabolite Daunorubicinol (DOL) in AML patients receiving induction chemotherapy and compared PK parameters with clinical outcome (relapse). Sixty four adult denovo AML patients were enrolled in the study after obtaining informed consent. Dnr was given as 1 hour bolus infusion at a dose of 60mg/m2/day for the first 3 days of induction chemotherapy. Peripheral blood were collected at 0, 15min, 1hr, 2hr, 4hr, 6hr and 24hr on the first day of induction chemotherapy. Plasma levels of Dnr and DOL were measured using high performance liquid chromatography coupled with fluorescence detector. Population PK was analyzed by nonlinear mixed effects modeling using Monolix version 4.3.2. Common single nucleotide polymorphisms in ABCB1, ABCG2, CBR1 and CBR3 genes were screened by PCR followed by either sequencing or restriction digestion. In vitro cytotoxicity of the primary AML cells to Dnr was assessed by MTT cell viability assay and IC50 was calculated using ADAPT5 software. The role of plasma Dnr PK on relapse of disease was estimated using nonparametric Mann-Whitney U test. Demographics of the patients enrolled in the study are listed in table 1. PK parameters of Dnr and DOL showed wide inter-individual variation (table2). Older patients had a trend towards lower Dnr clearance (CL) (p=0.09). Patients harboring FLT3-ITD mutations had higher CL than the patients without mutation though not reaching statistical significance. Patients with variants of SNPs rs20572 (exon3) and rs9024 (3’UTR) in CBR1 (which were in complete linkage disequilibrium) had a trend towards significantly higher Dnr AUC [median-0.2711; (0.04903-2.165) vs. median 0.2117; (0.0546-0.9036); p=0.06] than patients with wild-type. Among the 64 patients, complete remission (CR) was documented in 42 (65%) of which, 31% relapsed eventually [median time to relapse 328 (104-864) days]. AML patients who relapsed had significantly lower Dnr AUC [median 0.17 (0.049-2.165) vs. median 0.31 (0.1-1.74); p=0.009] and higher DOL AUC [median 8.67 (1.6-20.1) vs.4.99 (1.3-16.5); p=0.015].Also, relapsed patients had higher Dnr clearance (CL)[median -546.54 (41.5-1632)vs. 329.3 (42.9-944.5); p=0.011] and lower DOL CL[median-9.8 (4.9-56)vs. median- 19.7 (6.1-57); p=0.015]than patients who did not relapse. IC50 of Dnr was higher in patients who relapsed (median-0.7 (0.02-1.46) vs. median- 0.23 (0.007-1.11); p=0.056) than in patients who did not relapse. To the best of our knowledge, this is the first population PK report in AML which is designed to evaluate various factors which might influence Dnr and DOL PK in AML. Though the number is small, our results suggest that plasma PK of Dnr influences clinical outcome in AML. Lower systemic exposure of Dnr in relapsed patients suggests that amount of drug required to completely eradicate the leukemic cells is not achieved and escalating dose might be an option to decrease relapse rates. Cellular pharmacokinetics of Dnr in these patients is ongoing in the lab, which will be more informative and help in prognosticating and dose adjustment for better treatment outcome. This data provides a probable biological basis for the previously reported clinical benefit of decreased relapses following the increase of the dose of DNR in induction to 90mg/m2/day (Tallman et al. NEJM 2009).Wide inter-individual variation in Dnr PK also suggests that this increased dose may not be necessary for all patients. Further studies are required to confirm this finding in a larger cohort of patients and to work towards strategies for TDM of Dnr in AML Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Srivastava: Octapharma: Consultancy, Other.

Acute myeloid leukemia (AML) is a clinically and biologically complex and heterogeneous hematopoietic neoplasm. Recent advances in acute myeloid leukemia (AML) biology have lead to prognosticate and predict treatment outcome in AML based on molecular markers. Mutations in NPM1, CEBPA are considered good prognosis and High BAALC, ERG & MN1 expression associate with worse outcome in AML patients treated with standard chemotherapy. Although many efforts have been made to identify genetic mutations and modulated gene expression levels that can be used to predict outcomes in patients with AML, the association between these prognostic markers has not been evaluated. We have reported previously that the NPM1 mutated patients have significantly high dCK and hENT1 gene expression (involved in cytarabine metabolism) and low ABCG2 and ABCB1transporter expression (Abraham et al, ASH abstracts; Nov 2011; 118: 3481 and Nov 2012; 120: 143), suggesting that the good prognostic nature of this mutation is possibly due to the better metabolism and transport of the chemotherapeutic drugs used in induction therapy. We extended this study to look for association between NPM1/FLT3 mutation status and the RNA expression of other good or poor prognostic markers in patients with AML. We prospectively included 274 adult patients with AML in this study. The median age was 42 years (range 16-74y). AML was diagnosed according to the FAB and WHO classifications. There were 238 patients with de novo AML; Secondary AML -6; Therapy related AML- 2 and Relapsed AML-28. Bone marrow cytogenetics and immunophenotyping analysis was available for all patients at diagnosis and/or relapse. Diagnostic bone marrow MNCs were isolated by ficoll- density gradient centrifugation and stored in trizol reagent for RNA expression and mutation detection. RT-PCR was used to screen AML-ETO and Inv 16, and the expression of BAALC, ERG1, MN1, CXCR4 and WT1were analyzed using RQ-PCR. NPM1-c, FLT3 ITD and TKD were screened using DNA PCR followed by gene-scan, sequencing or RFLP methods. The basic demographics and the frequency of the markers are listed in Table 1. When analyzed separately in normal karyotype AML (NK-AML), the frequencies of the mutations were: NPM1: 52.2%; FLT3-ITD: 24%; TKD: 4.3%. When the RNA expression of BAALC, WT1, ERG1, CXCR4 and MN1 was compared in patients with NPM1 or FLT3 mutations, we noticed that patients with NPM1 had significantly low expression of BAALC, MN1 and ERG1 while those with FLT3 mutations (ITD or TKD) had higher expression of these genes (Figure1). There was no significant association with CXCR4 or WT1 expression and these mutations. When analyzed separately in the normal karyotype AML, these associations were still significant. In addition, the relapsed patients had significantly higher expression of BAALC, MN1, and ERG1 RNA compared to de-novoAML cases (data not shown). To conclude, we show that NPM1 or FLT3 mutations acquire the prognostic significance due to several factors including BAALC, ERG1 and MN1expression levels in addition to drug metabolizing enzymes’ and drug transporter expression. These factors must be taken into consideration when attempting to personalize chemotherapy in AML. Disclosures: No relevant conflicts of interest to declare.

Nuclear Hormone Receptors (NHRs) are a large superfamily of ligand dependent transcription factors regulating a plethora of genes involved in metabolism, growth and differentiation. Recent studies identified the overwhelming role played by NHR in determining hematopoiesis as well as HSC function and fate, suggesting that defective proliferation and maturation of progenitors in hematopoietic malignancies could be attributed to altered expression of these transcriptional regulators. NHRs are attractive but relatively unexplored drug targets and there is limited data on their expression profile in hematological malignancies. Our preliminary screening of AML cell lines and primary cells revealed differential expression of PPARg and RXRα among AML cell lines and decreased expression of these targets in resistant cell lines vs. sensitive ones (Fig.1a). We extended this study aiming to identify the expression pattern of all NHRs in myeloid malignancies, compare with ex-vivo chemoresistance and prognostic markers. Myeloid leukemia cell lines AML(n=12) carrying AML-ETO, Inv-16, PML-RAR fusion transcript, NPM1 or FLT3 mutation and CML(n=8), pooled primary samples harboring only FLT3 ITD mutation and only NPM1 mutation, CD34+ cells and total cellular RNA samples from normal donors were included in the study. Bone marrow samples from AML patients and peripheral blood samples from healthy volunteers were collected after informed consent. PBSC from normal donors (n=7) were enriched for CD34+ cells by magnetic enrichment. RNA extraction followed by purification and cDNA synthesis were done as per manufacturer’s recommendation. The expression of 42 NHRs and 42 co-regulators was profiled by qRT PCR using RT2 PCR Profiler array (SABiosciences, Germany). Expression of each gene was normalized to the average of 5 housekeeping genes (GAPDH, b-actin, b2-microglobulin, HGPRT & RPLP0) and differential expression was calculated by normalizing this with normal total RNA samples to determine fold change. Data normalization and differential gene expression were computed using SABiosciences web-based analysis software. Ex-vivo cytotoxicity to cytarabine and daunorubicin was analyzed using MTT assay and IC50 was calculated using Adapt software. Based on Ara-C and Dnr IC50, AML samples were categorized as sensitive or resistant (IC506.25µM for Ara-C;0.5µM for Dnr). FLT3-ITD and NPM1 mutations were screened using PCR followed by GeneScan methods. Upon analysis we observed steroid receptors except PPARG, NR5A1, RXRG, NR1D2, glucocorticoid receptors, androgen receptors and their co-regulatory molecules were expressed across all cell lines. In contrast, expression of certain NHRs such as NR2E3, NR1I2, NR1H4, NR0B1, NR0B2, ESRRG, ESRRB, NR1D1 and the co-regulator HDAC7 were less or at undetectable levels, while estrogen receptors and its co-regulatory molecules except PPARGC1B, steroid receptors HNF4A, NR2F1, NR2F2, RARG and NR3C2 were moderately expressed across the cell lines (Fig.1b). Analysis also revealed significantly reduced expression of ESR2, ESRRG, NR2F2, NR2F6 and THRB and increased PPARG in AML compared to CML cell lines. Comparison between CD34+ progenitor population and well differentiated hematopoietic cells demonstrated >6 fold change in NR4A1 expression. Likewise, expression of several differentiation markers NR1H3, NR1D2, RORA, RORB, RARA and VDR were elevated in cells with increased degree of maturation. The receptor inducing terminal differentiation of erythrocytes THRA, had lesser expression ( Our comprehensive analysis of NHRs and its co regulatory molecules provides us with insights on NHR expression on differentiation and drug resistance across myeloid leukemia cell lines with various genetic and molecular characteristics. This could be further explored to identify potential novel drug targets to be used as combination therapy in myeloid leukemias to overcome drug resistance. Disclosures: No relevant conflicts of interest to declare.

Wide inter-individual variation in terms of treatment outcome and toxic side effects of treatment exist among patients with AML receiving chemotherapy with cytarabine (Ara-C) and daunorubicin. We have previously evaluated the expression of the major genes involved in cytarabine transport and metabolism on ex-vivo Ara-C response and compared it with cytogenetic and molecular markers in AML (Blood (ASH Annual Meeting Abstracts) 2011 118: Abstract 3481). Our candidate gene expression data led us to propose Ara-C resistance index (Ara-C RI) (RI = ΔCT (DCK X ENT1)/ ΔCT CDA), which incorporates candidate Ara-C metabolizing genes whose RNA expression are significantly associated with ex-vivo Ara-C cytotoxicity. Ara-C RI values were significantly higher in resistant (IC50 >80 uM) and intermediate (IC50 6.25-80uM) samples when compared to sensitive samples (IC50 Though, the Ara-C RI correlated well with ex-vivo cytotoxicity as well as treatment response in vivo (data not shown), this did not completely explain the variation, suggestive of alternative resistance mechanisms. We undertook a genome-wide gene expression profiling to address possible mechanisms of Ara-C resistance other than the candidate gene approach. Based on ex vivo Ara-C cytotoxicity at diagnosis, Ara-C sensitive (IC50 80uM) (each 5) were included for microarray analysis. Total RNA was extracted from bone marrow mononuclear cells using tri reagent, cDNA was synthesized and then subjected to one color 8 X 60K Agilent microarray analysis. Data was normalized, filtered and analyzed using Gene Spring GX (V 12.0) software. Normalization was done using the 75th percentile shift (Percentile shift normalization is a global normalization, where the locations of all the spot intensities in an array are adjusted). Using unpaired t-Test, 4436 genes were identified to be differentially expressed (Fold change expression values were provided as log-base 2) between Ara-C sensitive samples and Ara-C resistant samples. Differentially regulated genes were clustered using hierarchical clustering based on Pearson coefficient correlation algorithm to identify significant gene expression patterns. Genes were classified based on functional category and pathways using GeneSpring GX and Genotypic Biointerpreter-Biological Analysis Software. The differentially expressed genes fell into the following biological processes; transcription (375 genes), transport (364 genes), metabolism (267 genes; Fig 1d), immune (155 genes), cell cycle (129 genes), apoptosis (123 gene; Fig 1c) and so on. Upregulated gene list in Ara-C sensitive group included apoptotic related genes like PMAIP1, NDUFA, BAX, BCL2, TRAF2, transcriptional regulators including SMAD5, SMAD1, ZNF family proteins- ZNF644, ZNF469, ZNF195, ZNF22, ZNF3, ZNF713, ZNF777, ZNF234, CEBPa, PARP, ETV6, E2F3 and so on. Down-regulated genes in Ara-C sensitive group are of special interest as they could be potential candidates for targeting Ara-C non-responsive group. They included transcriptional regulators like HDAC4, KLF4, CREB5, CEBPb, RARA, E2F4, MNDA, MTA3 and PPARd also apoptotic genes like MCL1, PSEN1, ELMO2, PAK1, APAF1, MAPK1, CD40, FAS, CASP1 and CASP8. Interestingly, many of the genes involved in cellular metabolism were found to be down regulated in AraC sensitive group. Major down-regulated metabolic genes include CDA, SLC2A3, SLC2A8, GK, NADK, ACACB, ACSL1, PFKFB4, PFKFB3, PDK4, ME1 and PC. This study has come up with previously unrecognized aspects in Ara-C resistance in AML, and is suggestive of Ara-C sensitive samples having increased expression of anti-Warburg set of genes. Our data as well as growing evidences from various malignancies and altered cellular metabolism propose the possibility of using metabolic inhibitors alone or in combination with Ara-C to overcome drug resistance. Disclosures: No relevant conflicts of interest to declare.

Abstract 1438 Combination chemotherapy in Acute Myeloid Leukemia (AML) can cure approximately 25% of patients but the majority is still non-responsive. Drug resistance and relapse are considered to be the major reasons for treatment failure. Though overexpression of ATP-Binding-Cassette (ABC) transporters including ABCB1 and ABCG2 have been shown associated with lower remission rates and survival in AML, the role of majority of ABC transporter genes are still unknown. Present study aims to determine the role of candidate ABC transporter RNA expression (that are shown to directly or indirectly influence sensitivity towards Cytarabine (Ara-C) or Daunorubicin (Dnr)), on in vitro cytotoxicity and the influence of prognostic markers (NPM, FLT3-ITD, AML-ETO, CBFB-MYH11 and CD34) on these transporter expression. 177 adult AML patients (median age: 43 years (16–74 years)) other than AML-M3 were included in this study. Total RNA was extracted from diagnostic bone marrow mononuclear cells and expression levels for each target gene (ABCB1, ABCB6, ABCB10, ABCA3, ABCA5, ABCA6, ABCC3, ABCC11 and ABCG2) relative to housekeeping gene GAPDH was analyzed using Taqman based gene expression assays. ABCB6 and ABCC3 assays were custom designed and were normalized to ABL as housekeeping gene. The dCT values (where dCT = CT value of target gene – CT value of housekeeping gene; lower dCT corresponds to higher expression and vice versa) were used for comparing expression between groups. In vitro cytotoxicity was assessed using MTT cell viability assay and IC-50 was calculated. Based on Ara-C and Dnr IC50, these samples were categorized as sensitive or resistant (IC50 5.2 uM for Ara-C; 0.5uM for Dnr). FLT3-ITD and NPM mutation status at diagnosis were determined by PCR followed by Genescan analysis using genomic DNA samples. AML-ETO and CBFB-MYH11 status were determined by RT-PCR. Wide variation in ABC transporter RNA expression was seen among AML patients (Fig 1a). Both ABCC3 and ABCB6 RNA expression were significantly higher (median ABCC3 dCT= 7.68 (−1.80–15.97) vs 10.62 (−0.66– 17.77); p= 0.0005; median ABCB6 dCT= 5.0 (−0.28 – 8.25) vs 5.4 (1.76 – 12.17); p=0.03)) in Dnr resistant patients (n=65) when compared to those who are sensitive to Dnr (n=53) (Fig 1b). No association was seen between any of these transporter expression and Ara-C in vitro cytotoxicity. Effects of NPM and FLT3 mutation status on ABC transporter genes were then evaluated. ABCB1 expression was significantly lower and ABCA5 expression was higher in patients with NPM mutation (n=50) when compared to those without mutation (n=126)-Table1. Patients with FLT3-ITD mutation (n=26) had a significantly lower ABCB1 expression when compared to those without mutation (n=150)-Table1. Low ABCB1 in this group could probably be due to the fact that NPM mutation was also positive in 16 out of 26 FlT3-ITD mutated patients. ABCB6 expression was significantly lower in FLT3-ITD positive patients (Table1). Other mitochondrial transporters like ABCG2 and ABCB10 showed a trend to low expression in FLT3-ITD patients when compared to those without FLT3 mutation (p=0.06 and p=0.1 respectively, Table1). Eighteen patients were positive for AML-ETO (n=12) or CBFB-MYH11 (n=6) and the RNA expression of ABCA5, ABCA3 and ABCG2 were significantly lower in this group compared to those who were negative for AML-ETO or CBFB-MYH11 (Table1). In addition, ABCB1 and ABCG2 expression was significantly higher in CD34 positive patients (CD34 >20% as analysed by flow cytometry) when compared to those with low CD34 expression (Table1) This study has come up with previously unrecognized aspects in ABC transporter expression and its role in AML. Low expression of ABCB1, a well accepted candidate for drug resistance, in NPM mutated AML could be one of the reasons which make NPM mutation a favourable prognostic marker. ABCC3 and ABCB6 which have shown association with Dnr sensitivity in this study could be a key avenue for further investigation. This comprehensive analysis on ABC transporter expression and its association with in vitro cytotoxicity and prognostic markers suggests ABCC3, ABCB6 and ABCA5 as probable targets which can be modulated for improving chemotherapeutic responses. Disclosures: No relevant conflicts of interest to declare.

Abstract 2484 Treatment failure in AML can be attributed to resistance to chemotherapeutic drugs. Induction chemotherapy of AML involves combination of Daunorubicin (Dnr) and Cytarabine. Up-regulation in the expression of efflux transporters such as ABCB1 and ABCG2 has been shown to be one of the causes of Dnr resistance in AML. Apart from efflux transporters, increased expression of the Dnr metabolising enzymes (Carbonyl reductase 1 (CBR1) and CBR3) also can influence the cytotoxic activity of Dnr against leukemic cells. Aim of the present study is to investigate: i) the role of mRNA expression of Dnr metabolising enzymes (CBR1 and CBR3) on the in vitro cytotoxicity of Dnr in primary AML cells and ii) effect of Single nucleotide polymorphisms (SNPs) in CBR1 and CBR3 on the plasma pharmacokinetics of Dnr and Daunorubicinol (DOL) in AML patients. Ninety patients with AML (excluding AML-M3) at diagnosis before the initiation of therapy were included. Bone marrow (BM) was collected at diagnosis and blast percentage in our cohort of patients ranged from 20–100%. Mononuclear cells (MNCs) were isolated by density gradient centrifugation. Total RNA was extracted from BMMNCs and cDNA was prepared. CBR1 and CBR3 mRNA expression was measured using Taqman based quantitative RT-PCR normalized to GAPDH. In-vitro cytotoxicity of Dnr was determined by MTT cell viability assay. Intracellular levels of Dnr and DOL was estimated by HPLC coupled with Fluorescence detector (FD). SNPs in CBR1 and CBR3 were screened by PCR followed by sequencing. In addition, plasma samples during Dnr infusion was collected at 0, 1, 2, 4, 6 and 24hrs from 24 of the 90 AML patients. Levels of Dnr and DOL in the plasma samples were estimated by HPLC coupled with FD and Area under Curve (AUC) for Dnr and DOL were calculated. CBR1 (range: 0.02–253.15) and CBR3 (range: 8.24– 29296.67) mRNA expression showed wide inter-individual variation. Intracellular levels of Dnr (range: 17.98–1718ng/3×106cells) and DOL (range: 2.76–62.07ng/3×106cells) showed 95 and 22 fold variation respectively. IC50 of Dnr in these samples ranged from 0.05 to 3.15 μM. Based on the IC50, samples were categorized as sensitive (IC501.0μM) to Dnr. CBR1 expression was significantly higher in resistant samples (median-102.99: range: 7.18–253.15) compared to intermediate (median- 29.73: range: 0.02–224.23) and sensitive samples (median-18.67: range: 0.34–105.40 p=0.0154) [Figure 1]. In addition, significant correlation between CBR1 expression and intracellular DOL levels was observed (r= 0.52; p Five SNPs in CBR1 and 3 in CBR3 were identified. CBR1 SNPs, rs20572, rs9024 and rs25678 were in complete linkage disequilibrium (LD). Variant of rs25678 and rs20572/rs9024 had an allelic frequency of 0.244 and 0.20 respectively. Among CBR3 SNPs, variants of two non-synonymous polymorphisms rs8133502 and rs1056892 showed allelic frequency of 0.31 and 0.51 respectively. AUC of Dnr and DOL in plasma samples ranged from 22–2474ng*h/ml and 37–2027 ng*h/ml respectively while clearance of Dnr ranged from 0.04 to 3.34 L/h. Plasma DOL AUC, unlike in vitro intracellular DOL, did not show any significant association with CBR1 and CBR3 expression. In patients with variant rs25678 genotype, Dnr AUC was higher and clearance was lower compared to the wild type genotype, reaching a trend to statistical significance (p=0.0537 and 0.1184 for Dnr AUC and clearance). Influence of Dnr and DOL plasma levels on clinical outcome, if any, remains to be evaluated. This is the first report showing the influence of CBR1 and CBR3 in in-vitro cytotoxicity of Dnr and role of SNPs in CBR1 and CBR3 in plasma pharmacokinetics of Dnr in AML patients. To conclude, higher expression of CBR1 confers resistance to Dnr in primary AML cells and polymorphisms of this gene may influence the pharmacokinetics of Dnr. Disclosures: No relevant conflicts of interest to declare.