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Drug resistance and relapse are common challenges in acute myeloid leukemia (AML), particularly in an aggressive subset bearing internal tandem duplications (ITDs) of the FLT3 receptor (FLT3-ITD+). The tyrosine kinase inhibitor gilteritinib is approved for the treatment of relapse/refractory AML with FLT3 mutations, yet resistance to gilteritinib remains a clinical concern, and the underlying mechanisms remain incompletely understood. Using transcriptomic analyses and functional validation studies, we identified the calcium-binding proteins S100A8 and S100A9 (S100A8/A9) as contributors to gilteritinib resistance in FLT3-ITD+ AML. Exposure of FLT3-ITD+ AML cells to gilteritinib increased S100A8/A9 expression in vivo and in vitro and decreased free calcium levels, and genetic manipulation of S100A9 was associated with altered sensitivity to gilteritinib. Using a transcription factor screen, we identified the transcriptional corepressor BCL6, as a regulator of S100A9 expression and found that gilteritinib decreased BCL6 binding to the S100A9 promoter, thereby increasing S100A9 expression. Furthermore, pharmacological inhibition of BCL6 accelerated the growth rate of gilteritinib-resistant FLT3-ITD+ AML cells, suggesting that S100A9 is a functional target of BCL6. These findings shed light on mechanisms of resistance to gilteritinib through regulation of a target that can be therapeutically exploited to enhance the antileukemic effects of gilteritinib.

To the editor: The human organic cation transporter (OCT1) has been implicated in the uptake of imatinib into chronic myeloid leukemia (CML) cells.[1][1][⇓][2][⇓][3]-[4][4] Moreover, OCT1 expression or activity is thought to be a critical determinant of imatinib response and is considered to

Background: Hydroxyurea has emerged as the primary disease-modifying therapy for both children and adults with sickle cell anemia (SCA). New guidelines from the National Heart, Lung, and Blood Institute include a recommendation that all children with SCA be offered hydroxyurea as young as nine months of age, regardless of the frequency or severity of clinical complications. The benefits of hydroxyurea are most evident when the dose is escalated to the maximum tolerated dose (MTD); however, a stable MTD may take 8-12 months to achieve. In this era of personalized medicine, individualized pharmacokinetics (PK) profiles can be used to predict appropriate medication dosing. Accordingly, we investigated hydroxyurea PK data in a large cohort of children with SCA at their first oral dose and at MTD, to understand inter-patient variability and to create an accurate dose prediction model for hydroxyurea MTD. Methods: The Hydroxyurea Study of Long-term Effects (HUSTLE, [NCT00305175][1]) is a longitudinal observational study for children with SCA receiving hydroxyurea therapy. Prospective investigation of hydroxyurea pharmacokinetics, pharmacodynamics, and pharmacogenomics is a primary study objective. HUSTLE includes two cohorts of children: a “New Cohort” with children initiating hydroxyurea therapy upon enrollment in the study, and an “Old Cohort” with children already taking hydroxyurea upon study entry. Children in the “New Cohort” received their first oral dose of hydroxyurea 20 mg/kg with formal first-dose PK data collection using peripheral blood analysis at baseline (t=0) followed by 15, 30, 60, 120, 240, and 480 minutes. PK analyses were performed at MTD for both the New and Old Cohorts. Hydroxyurea concentration was determined by a previously described color-based method, testing each sample in triplicate with a standard curve ranging from 0 to 1000mM, and sensitivity as low as 8mM. Results: A total of 97 children with SCA who enrolled in the New Cohort had first-dose hydroxyurea PK analysis, 65 of whom also had a paired PK study at MTD. Another 34 samples from the Old Cohort had PK analysis performed at MTD. | Patient samples | PK analysis timepoint | # of patients | Tmax (hr) | Cmax (mg/mL) | CL/F (L/hr) | t ½ (hr) | AUC (mg¥hr/mL) | | --------------- | --------------------- | ------------- | -------------------------- | -------------------------- | -------------------------- | -------------------------- | -------------------------- | | All | Baseline | 97 | 0.82 ± 0.47 (57.1) | 26.0 ± 6.60 (25.4) | 6.77 ± 3.14 (46.4) | 1.64 ± 0.55 (33.3) | 91.8 ± 23.0 (25.0) | | All | MTD | 99 | 0.80 ± 0.52 (65.1) | 31.2 ± 8.08 (25.9) | 7.27 ± 2.99 (41.2) | 1.94 ± 0.87 (44.6) | 115 ± 24.1 (20.9) | | Paired | Baseline | 65 | 0.82 ± 0.46 (56.4) | 25.3 ± 5.56 (22.0) | 7.11 ± 3.16 (44.4) | 1.73 ± 0.49 (28.5) | 93.1 ± 23.1 (24.8) | | Paired | MTD | 65 | 0.74 ± 0.47 (62.8) | 31.0 ± 7.82 (25.2) | 7.40 ± 3.13 (42.3) | 1.82 ± 0.89 (48.9) | 114 ± 22.3 (19.5) | Abstract 2707. Table 1. Baseline (first-dose) hydroxyurea pharmacokinetics revealed substantial interpatient variability, with a coefficient of variation ranging from ~25% for maximum concentration (Cmax) and area-under-the-curve (AUC) to ~50% for time to maximum concentration (Tmax) and apparent oral clearance (CL/F). At MTD, the PK parameters showed an expected increase in Cmax due to the higher average dose, but a convergence toward AUC of ~115 (mg¥hr/mL) with a reduced coefficient of variation. Based on this target AUC, predictive equations were generated that include PK parameters, but the simplest equation for the starting hydroxyurea dose is: Dose (mg) = 400 – [1000 x creatinine] + [21 x weight]. Conclusions: Individual hydroxyurea absorption patterns for young children with SCA can be used to predict MTD. The starting dose can be optimized by using predictive equations that reflect apparent oral clearance instead of body weight alone, and subsequently titrated by marrow suppression. AUC is the most relevant PK parameter, and our data show its variability is consistently lower at MTD, with a target AUC of 115 mg.hr/mL. As the use of hydroxyurea therapy expands, we plan a prospective study to tailor the starting dose using a pharmacometrics model involving weight, renal function, and selected PK parameters to rapidly achieve a safe and stable MTD. Disclosures Off Label Use: Hydroxyurea use for children with sickle cell anemia. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00305175&atom=%2Fbloodjournal%2F124%2F21%2F2707.atom

Abstract 1008 Background: Sickle cell is the most common inherited blood disorder affecting millions worldwide. Hydroxyurea is currently the only FDA approved drug for the treatment of sickle cell anemia (SCA) that alters disease pathophysiology. Known for inducing fetal hemoglobin production, hydroxyurea can decrease hospitalizations, vaso-occlusive pain episodes, and mortality associated with SCA. Although efficacious for many patients, a large degree of inter-patient variability in pharmacokinetic (PK) and pharmacodynamic (PD) properties of hydroxyurea has been noted, but sources of this variation remain largely undefined. Plasma transmembrane transporters have been shown to play an important role in the determination drug PK and PD of various xenobiotics. Our recent in vitro studies have demonstrated that hydroxyurea is a substrate for specific solute carrier (SLC) transporters including members of the organic anion transporting polypeptide 1B (OATP1B) subfamily which consists of OATP1B1, and OATP1B3 in humans, and one mouse orthologue Oatp1b2. These transporters are highly expressed on the sinusoidal membrane of hepatocytes, and function as uptake transporters bringing substrates from the blood into the liver. Limited reports have suggested that Oatp1b2 transcript is also found in kidney and intestine as well. In the present study we evaluated the uptake of the hydroxyurea by different haplotypes of hOATP1B3 transporter in vitro, as well as the influence of the Oatp1b2 transporter on hydroxyurea PK in vivo using a transporter knock-out mouse model. Methods: Transporter-mediated cellular uptake of hydroxyurea was determined in vitro by measuring [14C]-hydroxyurea accumulation in Xenopus laevis oocytes that over express two common haplotypes of OATP1B3. hOATP1B3*1 and the functional variant hOATP1B3*2 that contains the 334T>G and 699G>A SNPs were assessed. In vivo PK Studies were performed using a knockout model mouse model in which mOATP1b2 was eliminated from the DBA/LcaJ1 background. Mice were dose with 50mg/kg of [14C]-hydroxyurea via oral gavage or intravenous (i.v.) injection. Serial blood samples were collected at defined timepoints and concluded with terminal blood and tissue collections at 2 hours post injection. Hydroxyurea levels were determined based on radioactivity in plasma and liver, and kidney homogenate. Accumulation in the urine was also measured over a 3 day period. Results: In vitro kinetic studies did not show any differences between OATP1B3*1 and OATP1B3*2 haplotypes in time-, or concentration-dependent accumulation of hydroxyurea. The inhibitor substrates naringen and rifampin decreased hydroxyurea accumulation by at least 50% in OATP1B3*1 but did not decrease accumulation in OATP1B3*2. These in vitro studies suggest that the genetic mutations associated with these two haplotypes do not have a functional impact on the transport of hydroxyurea, but may have significant implications for potential drug-drug interaction. In vivo, Hydroxyurea PK was altered in the absence of the Oatp1b2 transporter. Plasma levels of hydroxyurea were similar after i.v. injections, but total exposure (AUC) in Oatp1b2-KO mice was decreased by 20% with an AUC of 2257 mg-min/ml compared to 2872 mg-min/ml observed in the wildtype mice (p=0.04). Hydroxyurea levels in the kidneys of Oatp1b2-KO mice were approximately 50% of wildtype with a mean accumulation of 360 pmol/g tissue (p= 0.0025). In addition, 30% less hydroxyurea was excreted in urine of Oatp1b2-KO mice by 72 hours (p=0.023). Accumulation of hydroxyurea in liver of Oatp1b2-KO mice and wildtype mice were 107 and 132 pmol/g tissue respectively, but this difference was not statistically significant. These results suggest that hydroxyurea PK is altered in the absence of Oatp1b2 transporter after oral administration. Conclusions: These studies provide the first in vivo evidence that specific transporters may be key determinants of hydroxyurea PK. Future studies are warranted to investigate the influence of human OATP1B and other transporters that modulate hydroxyurea PK. Identification of such transporters may help to elucidate factors such as genetic variations in the transporters and/or drug-drug interactions that may contribute to PK and PD variability observed in sickle cell patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 2131 Background: Over the past 15 years, numerous prospective and cross-sectional clinical trials have demonstrated that hydroxyurea has both laboratory and clinical efficacy for pediatric patients with sickle cell anemia (SCA). In infants, toddlers, children, and adolescents, hydroxyurea administered at maximum tolerated dose (MTD) leads to significant increases in hemoglobin (Hb) concentration, MCV, and %HbF along with simultaneous decreases in neutrophils, reticulocytes, total bilirubin, and serum lactate dehydrogenase. Clinical benefits include reduction in acute vaso-occlusive events (pain and acute chest syndrome), and emerging data suggest protection against chronic organ damage with a low risk of genotoxicity. For individual patients, however, the %HbF response to hydroxyurea and the MTD dose itself are highly variable. Currently there are no accurate predictors of the final %HbF or the MTD dose. Methods: To address the phenotypic variability, the Hydroxyurea Study of Long-Term Effects (HUSTLE, NCT00305175) was designed to capture prospectively first-dose (20 mg/kg) pharmacokinetics (PK) of hydroxyurea including maximum serum concentration (Cmax), area under the concentration curve (AUC), apparent oral clearance (CL/F), half-life (t1/2), and apparent volume of distribution (V/F). A consistent dose escalation schedule was then used to achieve a stable MTD, at which time PK studies were repeated and hydroxyurea responses (%HbF and MTD dose) were recorded along with other pharmacodynamics parameters. Results: After written informed consent, a total of 98 pediatric patients commenced hydroxyurea, 65 of whom reached MTD with complete paired PK information. In the majority of patients (39 of 65), a ‘fast/slow’ absorption phenotype identified at first-dose PK analysis was retained at MTD, supporting the concept of a genetic basis for this variation. Inter-individual PK variability was substantially greater than intra-individual variability; for example, the coefficient of variation (%CV) for CL/F was 44.4% on day 1 and 42.3% at MTD among all subjects, but averaged only 12.8% within the same subject. The CL/F was partly dependent on the rate of absorption but was most strongly influenced by subject weight and serum creatinine. At MTD, the average AUC was 114 ± 22.3 mg·h/mL, and the %CV of AUC was reduced from 24.8% on first-dose PK to 19.5% at MTD, likely reflecting dose titration toward a common degree of myelosuppression. In an attempt to predict the MTD dose toward this target AUC using data available at baseline, ‘best-fit’ equations were developed such as the following with or without PK data: Predicted MTD (mg/kg/day) = 31.9 - [17.1*Baseline Creatinine] - [0.14*Baseline BMI] + [0.0036*Baseline ARC] Predicted MTD (mg/kg/day) = 32.6 - [15.1*Baseline Creatinine] - [0.16*Baseline BMI] - [0.56*First-dose half-life] + [0.0034*Baseline ARC] - [0.48*PK phenotype (fast=0, slow=1)] A simpler equation for predicting MTD dose using only baseline creatinine and weight was then developed: Predicted MTD (mg) = 400 - [1000*Baseline Creatinine] + [21*weight] Conclusions: The relatively small intra-individual PK variability at hydroxyurea MTD compared to baseline PK studies, coupled with a similar degree of drug exposure when patients achieve a stable MTD, supports future investigation to predict the optimal hydroxyurea dose prior to the first dosing. Prediction equations of the MTD should be tested prospectively against standard dose-escalation schedules. Disclosures: Off Label Use: Hydroxyurea for children with sickle cell disease.

Abstract 571 Methotrexate is an antifolate chemotherapeutic drug commonly used to treat cancer, including acute lymphoblastic leukemia (ALL). Interindividual variation in clearance of methotrexate results in a vast range of exposure to the drug, which affects its clinical effectiveness and risk of toxicity. In a genome-wide association study of children with ALL, we identified the SLCO1B1 gene, encoding liver transporter OATP1B1, as harboring multiple common polymorphisms associated with methotrexate clearance (Trevino et al. J Clin Oncol, 27:5972-5978, 2009). Because these common polymorphisms could not account for the variation in methotrexate clearance entirely, we hypothesized that there may be rare variants in addition to the common polymorphisms in this gene that affect methotrexate disposition. To test this hypothesis, we sequenced SLCO1B1 exons in 699 pediatric leukemia patients, and compared the effects of common versus rare variants on methotrexate clearance (adjusting for clinical covariates). We identified 93 SNPs, 15 of which were non-synonymous (NS); 11 of the 15 NS SNPs were rare, with minor allele frequencies < 1%. We used four computational programs (SIFT, PMUT, SNPS3D, and Polyphen2) to predict whether the NS SNPs were damaging to the function of the protein. The 7 NS SNPs predicted to be functionally damaging (common or rare) were more likely to be found among patients with the lowest methotrexate clearance than patients with high clearance (p=2.4×10−8), including one SNP that was observed only once among nearly 1400 chromosomes. Four SLCO1B1 haplotypes were associated with reduced methotrexate clearance (p Disclosures: Evans: St. Jude Children's Research Hospital: Employment, Patents & Royalties; NIH NCI: Research Funding; Aldagen: Membership on an entity's Board of Directors or advisory committees. Relling:Sigma-Tau Pharmaceuticals, Inc: Investigator-initiated research; St. Jude Children's Research Hospital: Employment, Patents & Royalties; NIH: Research Funding.

Abstract 2575 Poster Board II-552 Background: Hydroxyurea is the only FDA-approved drug for the treatment of sickle cell anemia (SCA) in adults. Hydroxyurea increases fetal hemoglobin, decreases hospitalizations and painful events, and reduces mortality. With an oral bioavailability of > 90%, hydroxyurea is rapidly absorbed and distributed throughout the body. Though hydroxyurea has proven to be effective in treating SCA, there is considerable inter-patient variability observed in the pharmacokinetics and pharmacodynamics of hydroxyurea. Currently, mechanisms involved in the absorption, distribution, and elimination of hydroxyurea remain unclear. Recently, key transmembrane proteins have been identified as drug transporters due to their ability to move a variety of xenobiotic substances across cell membranes. Drug transporters are widely distributed throughout the body, and most are specific to certain substrates. Solute carrier (SLC) transporters in particular have been to shown to significantly impact drug pharmacokinetics by influencing the absorption, distribution, and elimination of specific drugs. The present study was designed to identify SLC transporters that may influence the absorption, distribution, and/or elimination of hydroxyurea in patients with SCA. Methods: In vitro studies using an equilibrium dialysis plate were performed to determine the amount of hydroxyurea that binds to human serum proteins. Transporter-mediated cellular uptake of hydroxyurea was determined in vitro by measuring [14C]-hydroxyurea accumulation in HEK293 cells and oocytes that overexpress organic anion transporters (OAT1-3), organic cation transporters (OCT1-3), organic cation/carnitine transporters (OCTN1-2), organic anion transporting polypeptides (OATP1A2/OATP1B1/OATP1B3), or vector control. LLC-PK1 cells that overexpress urea transporters A or B (UTA/UTB) were used to determine UTA/UTB mediated transcellular transport of hydroxyurea in transwell plates. The transport of [14C]-hydroxyurea from apical to basal or from basal to apical compartments was measured for the UTA/UTB overexpressing cells and compared to vector control. UTA and UTB mRNA expression was measured by real-time PCR of cDNA obtained from human tissue samples. Results: Protein binding assays showed that >76% of [14C]-hydroxyurea remained unbound to proteins in human serum containing hydroxyurea at concentrations ranging from 1.5μM to 500μM. The fraction of unbound hydroxyurea was similar using serum obtained from pediatric patients with SCA. In uptake studies, [14C]-hydroxyurea was a potent substrate for OATP1B3 with an approximately 2-fold increase in drug accumulation compared to control (p Conclusion: Cellular uptake of hydroxyurea is mediated by active transport via specific SLC transporters OATP1B3, UTA and UTB, which are expressed in liver, kidney, brain, intestine, and blood cells. Studies to further characterize hydroxyurea transporters should improve our understanding of the pharmacokinetic and pharmacodynamic profiles of hydroxyurea used in clinical practice for patients with SCA. Disclosures: No relevant conflicts of interest to declare.

Hydroxyurea therapy is now used commonly for both adults and children with sickle cell anemia (SCA), and has both laboratory and clinical efficacy. At doses ranging from 10–35 mg/kg/day, biologically important laboratory responses to hydroxyurea include induction of fetal hemoglobin (HbF), increased hemoglobin concentration, decreased circulating white blood cell and reticulocyte counts, and reduction of hemolysis (lower LDH). Treatment with hydroxyurea at the maximum tolerated dose (MTD) provides optimal laboratory responses in children with SCA. Despite the proven efficacy of this therapeutic option, hydroxyurea has a variable effect among patients with SCA that is not well understood. Patients have variability in their HbF and LDH responses, but also in the toxicity profile, which typically manifests as myelosuppression and limits the MTD. To understand better the variation in hydroxyurea responses and toxicities, we performed first-dose pharmacokinetic (PK) studies on a cohort of children with SCA who were initiating hydroxyurea therapy for clinical indications. As part of the prospective IRB-approved research protocol Hydroxyurea Study of Long-term Effects (HUSTLE, ClinTrials.gov [NCT00305175][1]), 40 pediatric patients received a single 20 mg/kg oral dose of hydroxyurea (range 18.0–21.6 mg/kg) and had timed venous blood collection at 0, 30, 60, 120, 240, and 480 minutes. Serum was assayed for hydroxyurea concentration using a colorimetric chemical assay. Estimates of PK parameters for hydroxyurea were derived from individual concentration-time data sets using model independent methods. Maximum plasma concentration (Cmax) and the time of maximum concentration (Tmax) were observed; area under the concentration-time curve (AUC) was calculated using the final quantifiable sample and also by extrapolation to infinity. Additional PK parameters included terminal half-life (t1/2), apparent volume of distribution (V/F), and apparent oral clearance (CL/F). For all 40 patients, the apparent oral clearance (CL/F) of hydroxyurea was 0.252 ± 0.080 L/hr/kg, which is slightly increased compared to previously published values for adults. Hydroxyurea was rapidly absorbed after oral administration with a median time to peak concentration of 0.55 hours (range, 0.467–2.2 hours). Interindividual PK variability of hydroxyurea was substantial; the coefficient of variation was 26.0% for t1/2, 30.3% for Cmax, 46.7% for CL/F, and 61.6% for V/F. Variability in apparent oral clearance was related primarily to patient weight; linear regression indicated R-squared value of 0.533 indicating that over half of the observed interindividual variability could be accounted by weight alone. There was no significant contribution from hepatic or renal function, including glomerular filtration rate. Two distinct PK profiles for hydroxyurea were observed when patients were grouped on the basis of time to peak concentration: rapid and slow absorption phenotypes. For 25 children, the peak hydroxyurea concentration was observed at the first sampling time point, suggesting very rapid absorption; using a linear one-compartment model the predicted median time to peak concentration was 18 minutes. In these patients with the rapid absorption profile, the median Cmax was 74% higher than for the slow absorption (P

Methotrexate is a major component in every treatment protocol for childhood acute lymphoblastic leukemia (ALL). Both beneficial and detrimental effects of methotrexate in ALL have been clearly related to methotrexate plasma pharmacokinetics, which vary substantially among patients. However the genetic basis of such variability remains largely unknown. Herein, we surveyed 600,000 germline single nucleotide polymorphisms (SNPs) to determine how inherited genetic variation affects the disposition of methotrexate among 434 children with ALL who received 3014 courses of methotrexate at 2 to 5 g/m2. Adjusting for age, race, gender and methotrexate regimen, the most significant SNPs associated with methotrexate clearance were annotated to a plausible gene, the organic anion transporter polypeptide, SLCO1B1. The three top SNPs included rs11045879 (P = 1.7 × 10−10), rs4149081 (P = 1.7 × 10−9), and rs2900478 (P = 2.8 × 10−8). Linkage disequilibrium (LD) was observed among these three SLCO1B1 SNPs (r2=1) and with a known functional polymorphism in SLCO1B1, T521C (rs4149056, r2 = 0.86). The top two SLCO1B1 SNPs rs11045879 and rs4149081 were further validated (P = 0.018 and P = 0.017) in an independent cohort of 206 patients with ALL. Additional SNPs annotated to SLCO1B1 were identified and further validated. In a stepwise multiple linear regression analysis, SLCO1B1 genetic variation remained significant and explained clearance variability comparable to that of other non-genetic factors including treatment regimen. SNPs in SLCO1B1 were also associated with methotrexate-related gastrointestinal toxicity (P= 0.03 to 0.0005, odds ratio 8.3 to 16.4). In summary, we have identified a candidate gene, SLCO1B1, which is strongly associated with the pharmacokinetics of methotrexate, an anticancer drug with a low therapeutic index in multiple treatment regimens. Although SLCO1B1 is widely recognized as having a strong impact on the disposition of many drugs in clinical use, based on in vitro data, it was not thought to have a major role in methotrexate transport or disposition. Our study demonstrates proof of principle that genome-wide tools in clinical pharmacologic problems can lead to the discovery of important and novel pharmacogenetic links between inherited genomic variation and drug response in humans.