Purpose:

OBI-3424 is a highly selective prodrug that is converted by aldo-keto reductase family 1 member C3 (AKR1C3) to a potent DNA-alkylating agent. OBI-3424 has entered clinical testing for hepatocellular carcinoma and castrate-resistant prostate cancer, and it represents a potentially novel treatment for acute lymphoblastic leukemia (ALL).

Experimental Design:

We assessed AKR1C3 expression by RNA-Seq and immunoblotting, and evaluated the in vitro cytotoxicity of OBI-3424. We investigated the pharmacokinetics of OBI-3424 in mice and nonhuman primates, and assessed the in vivo efficacy of OBI-3424 against a large panel of patient-derived xenografts (PDX).

Results:

AKR1C3 mRNA expression was significantly higher in primary T-lineage ALL (T-ALL; n = 264) than B-lineage ALL (B-ALL; n = 1,740; P < 0.0001), and OBI-3424 exerted potent cytotoxicity against T-ALL cell lines and PDXs. In vivo, OBI-3424 significantly prolonged the event-free survival (EFS) of nine of nine ALL PDXs by 17.1–77.8 days (treated/control values 2.5–14.0), and disease regression was observed in eight of nine PDXs. A significant reduction (P < 0.0001) in bone marrow infiltration at day 28 was observed in four of six evaluable T-ALL PDXs. The importance of AKR1C3 in the in vivo response to OBI-3424 was verified using a B-ALL PDX that had been lentivirally transduced to stably overexpress AKR1C3. OBI-3424 combined with nelarabine resulted in prolongation of mouse EFS compared with each single agent alone in two T-ALL PDXs.

Conclusions:

OBI-3424 exerted profound in vivo efficacy against T-ALL PDXs derived predominantly from aggressive and fatal disease, and therefore may represent a novel treatment for aggressive and chemoresistant T-ALL in an AKR1C3 biomarker-driven clinical trial.

Translational Relevance

In this study we investigated the efficacy of a novel prodrug, OBI-3424, in preclinical models of pediatric acute lymphoblastic leukemia (ALL). OBI-3424 is activated by the enzyme aldo-keto reductase 1C3 (AKR1C3) into a potent DNA-alkylating agent. Using a large cohort of primary patient samples and patient-derived xenografts (PDX) we showed that AKR1C3 expression was significantly higher in T-lineage ALL (T-ALL) than B-lineage ALL and almost all normal human tissues. In addition, OBI-3424 exerted potent in vitro cytotoxicity against T-ALL cell lines and PDX cells. When tested in vivo using PDX models in immunodeficient mice, OBI-3424 at drug exposure levels likely to be achieved in humans profoundly reduced the bone marrow infiltration of pediatric T-ALL PDXs, and significantly delayed disease progression. OBI-3424 also caused prolongation of mouse survival when combined with nelarabine in T-ALL PDXs and represents a potential novel treatment for aggressive and chemoresistant T-ALL.

Five-year survival rates for children diagnosed with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have continually improved since the 1970s and are currently approximately 90% (1–3). Pediatric ALL can be broadly divided into B-lineage ALL (B-ALL) and T-lineage ALL (T-ALL; ref. 4). Disease-free survival and overall survival are comparable between children with B-ALL and T-ALL treated on contemporary risk-adapted regimens (5), although outcome is poor for children with T-ALL following relapse (6).

Aldo-keto reductases (AKR) are a superfamily of NAD(P)(H)-dependent oxidoreductases that primarily catalyze the reduction of aldehydes and ketones to their corresponding alcohols (7). AKR family I member C3 (AKR1C3), is a hydroxysteroid dehydrogenase involved in the synthesis of steroid hormones and prostaglandins (8) and is expressed in a range of normal human tissues at varying levels (9). AKR1C3 is also expressed in pediatric and adult ALL, with significantly higher mRNA levels detected in T-ALL than B-ALL (10). Recently, elevated expression and activity of AKR1C3 and other subfamily members were shown in diagnostic samples from pediatric patients with T-ALL who went on to respond poorly to treatment when compared with treatment responders, suggesting a relationship between AKR1C3 expression and sensitivity of T-ALL cells to conventional drugs including daunorubicin, vincristine, and l-asparaginase (11). Moreover, the activity of AKR1C3 and its subfamily members affects the in vitro sensitivity of primary T-ALL cells and cell lines to vincristine (11). Therefore, AKR1C3 overexpression in pediatric T-ALL is a potential therapeutic target, as well as a possible biomarker for treatment resistance.

A potential innovative therapeutic approach is to exploit the enzymatic activity of AKR1C3 to activate novel prodrugs. The hypoxia-activated nitrogen mustard pre-prodrug, PR-104, is hydrolyzed in vivo to PR-104A, which then undergoes activation under hypoxic conditions to DNA cross-linking metabolites by 1-electron NADPH:cytochrome P450 oxidoreductase (CYPOR) and related flavoproteins (12, 13). While PR-104 was initially developed as a hypoxia-activated prodrug, it was subsequently discovered to also be activated by AKR1C3 under aerobic conditions (13). Despite exhibiting promising preclinical activity against T-ALL patient-derived xenografts (PDX; ref. 14), PR-104 showed limited efficacy in a phase I/II clinical trial in adult patients with ALL or acute myeloid leukemia, and myelosuppression was the major dose-limiting toxicity (DLT; refs. 15, 16). Moreover, PR-104 also exhibits considerable bystander effects in its mechanism of action, which is also likely to contribute to both its efficacy and toxicity (17).

ALL cells are known to be preferentially sensitive to DNA-damaging agents. The most effective DNA-alkylating agent used to treat pediatric ALL, cyclophosphamide, is itself a prodrug converted by liver enzymes to active metabolites that then enter the circulation to reach their target cells. Myelosuppression is also the major DLT of cyclophosphamide (18). Theoretically, a potent DNA-alkylating prodrug that is both activated and retained within the target cancer cell may exhibit favorable properties of safety, selectivity, and efficacy compared with both cyclophosphamide and PR-104. OBI-3424 (previously TH-3424) is a prodrug that is selectively activated by AKR1C3 to a potent DNA-alkylating agent that is then retained within the cell in which it is activated. It has entered clinical testing for hepatocellular carcinoma and castrate-resistant prostate cancer (NCT03592264). The goal of this study was to evaluate the efficacy of OBI-3424 against preclinical models of pediatric ALL with reference to AKR1C3 expression.

Cell lines and PDXs for in vitro and in vivo studies

The non–small cell lung cancer H460 cell line was purchased from ATCC. All cell line studies were outsourced to HD Biosciences. All experimental work was performed with approvals from the respective institutional review boards and animal ethics committees of each institution. Experiments used continuous PDXs established previously in 20–25 g female nonobese diabetic/SCID (NOD.CB17-Prkdcscid/SzJ, NOD/SCID) or NOD/SCID/IL2 receptor γ–negative (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJAusb, NSG) mice, as described elsewhere (19). The development of lentivirally transduced ALL-11 PDXs [empty vector (EV) and AKR1C3 overexpressing] has been described previously (10). The patient demographics and passage numbers of the ALL PDXs used in this study are shown in Supplementary Table S2, and genomic characterization can be accessed at https://pedcbioportal.org and in Supplementary Table S3. The ALL-11/EV and ALL-11/AKR1C3 PDXs were used at fifth passage. OBI-3424 was provided by Threshold Pharmaceuticals, Inc. and is being developed by OBI Pharmaceuticals, Inc., and Ascentawits Pharmaceuticals, Ltd.

In vitro cytotoxicity assays

H460 and leukemia cell lines (Supplementary Table S1) were suspended in RPMI medium supplemented with FBS (Biosera), whereas ALL PDXs cells were cultured in QBSF medium (Quality Biological Inc,) supplemented with Flt-3 ligand (20 ng/mL, BioNovus Life Sciences) or IL7 (10–20 ng/mL, Jomar Life Research). Cells were plated according to optimal cell density (14, 20) and incubated for 3 hours or overnight (37°C, 5% CO2). H460 cells were pretreated with 3 μmol/L TH-3021 (SN336384), a potent and specific inhibitor of AKR1C3 (21), for 2 hours, cotreated with OBI-3424 for 2 hours, washed, and incubated in fresh medium for 48 hours. PDX cells and leukemia cell lines were treated with OBI-3424 (10 μmol/L–1 pmol/L) or vehicle control for 48 or 72 hours, respectively. Viability was determined using Alamar Blue reduction assay (14, 22), or Cell Titer-Glo Luminescent Cell Viability Assay (Promega). The half-maximal inhibitory concentration (IC50) was calculated by interpolation of nonlinear regression curves calculated by GraphPad Prism 7 software.

RNA-Seq analysis

For analysis of AKR1C3 expression in primary patient aspirates, patients were stratified into B-ALL and T-ALL and their relevant subgroups as described previously (23, 24). Paired-end reads were mapped to the GRCh37 human genome reference by STAR (ref. 25; version 2.5.1b) through the recommended two pass mapping pipeline with default parameters, and the Picard MarkDuplicates module was used to mark the duplication rate. Gene annotation files were downloaded from Ensembl (http://www.ensembl.org/) and used for STAR mapping and subsequent gene expression level evaluation. To evaluate gene expression profiles, read counts for annotated genes were called by HTSeq (ref. 26; version 0.6.0) and processed by DESeq2 R package (27) to normalize gene expression into regularized log2 values.

For analysis of PDX samples, Illumina paired-end RNA-seq data were aligned to the human genome assembly (build hg38) using STAR (version 2.5) with quantMode parameters set to TranscriptomeSAM for alignments translated into transcript coordinates. Alignments were run through RSEM (version 1.2.31) command rsem-calculate-expression to calculate raw gene counts, TPM, FPKM, and isoform expression. All RNA-seq values are expressed as fragments per kilobase million (FPKM). FPKM data were log2 transformed.

Immunoblotting and qRT-PCR

Procedures for immunoblotting and qRT-PCR have been described previously (14, 28, 29) and are detailed in the Supplementary Materials and Methods, and data were quantified relative to HeLa cells.

Single-cell gel electrophoresis assays

Single-cell alkali gel electrophoresis to calculate the interstrand cross-link (ICL) index was performed using the Trevigen CometAssay Kit (Bio-Scientific) and is described in detail in the Supplementary Materials and Methods (30).

Activation and stability of OBI-3424 in mouse, monkey, and human cytosol, plasma, and liver microsomes

Samples of plasma, liver cytosol (1 mg/mL, in 100 mmol/L PBS, 2 mmol/L NADPH), and liver microsomes (0.5 mg/mL, in 100 mmol/L PBS, 2 mmol/L NADPH) were each obtained from mouse, monkey and human. For each sample, OBI-3424 was incubated at a final concentration of 5 μmol/L (plasma and liver cytosol) or 1 μmol/L (liver microsomes) at 37°C for 120 minutes (plasma), 60 minutes (liver cytosol), or 45 minutes (liver microsomes). Progesterone (5 μmol/L) and midazolam (1 μmol/L) were used as positive controls for liver cytosol and liver microsomes, respectively. In addition, liver cytosol samples were tested ±3 μmol/L TH-3021 (21). Reactions were terminated by adding acetonitrile containing propranolol as an internal standard. After centrifugation at 4°C, the supernatants were analyzed by LC/MS-MS.

Pharmacokinetic study of OBI-3424 in mice and monkeys

Procedures for the evaluation of OBI-3424 pharmacokinetics and toxicity in nude mice and cynomolgus monkeys are detailed in the Supplementary Materials and Methods.

Assessment of in vivo drug efficacy

Leukemia engraftment and progression were assessed in groups of 8 female 20–25 g NSG mice following intravenous (i.v.) inoculation of PDX cells by weekly flow cytometric enumeration of the proportion of human versus mouse CD45+ (%huCD45+) cells in the peripheral blood (PB). Individual mouse event-free survival (EFS) was calculated as the number of days from treatment initiation until the %huCD45+ reached 25%, computed by interpolating between bleeds directly preceding and following events, assuming log-linear growth. Efficacy of drug treatment was evaluated by the difference between median EFS of vehicle control (C) and drug-treated (T) cohorts, as well as T/C values, and by an objective response measure, as described previously (see Supplementary Materials and Methods; Supplementary Table S4; ref. 31). Leukemic infiltration was also assessed in the femoral bone marrow, spleen, and blood at day 0 (n = 3 mice), day 28 (n = 4 mice), or event, whichever occurred first. OBI-3424 (or vehicle control) was administered via intraperitoneal (i.p.) injection once weekly for 3 weeks. For combination studies, mice were treated with OBI-3424 administered on the above schedule plus nelarabine at 150 mg/kg via i.p. injection once daily for 5 days and repeated at day 14.

Statistical analysis

All statistical methods used in this study are described in detail in the Supplementary Materials and Methods.

Structure and in vitro antileukemic efficacy of OBI-3424

OBI-3424 (previously TH-3424) was developed as a highly potent DNA-alkylating prodrug that is selectively activated by AKR1C3 (Fig. 1A). In the presence of NADPH, OBI-3424 is reduced by AKR1C3 to an intermediate that spontaneously hydrolyzes to OBI-2660 (Fig. 1A), which has a structure reminiscent of the DNA-alkylating drug thioTEPA (N,N′,N″-triethylenethiophosphoramide; ref. 32). OBI-3424 exerted potent cytotoxicity against the H460 lung cancer cell line (IC50 4.0 nmol/L); in contrast the IC50 of OBI-2660 was >330 μmol/L (Fig. 1B). The cytotoxicity of OBI-3424 was highly AKR1C3 dependent (Fig. 1B); OBI-3424 IC50 values were 4.0 nmol/L and 6.3 μmol/L in the absence or presence, respectively, of 3 μmol/L TH-3021.

Figure 1.

In vitro activity of OBI-3424. A, Chemical structure and mechanism of activation of OBI-3424. B, Cytotoxicity of OBI-3424 against H460 cells ± TH-3021, a specific inhibitor of AKR1C3. Cytotoxicity of OBI-3424 against a panel of six B-ALL (C), seven T-ALL (D), and six ETP-ALL (E) PDXs. Data in CE are displayed as the mean ± SEM of biological triplicates.

Figure 1.

In vitro activity of OBI-3424. A, Chemical structure and mechanism of activation of OBI-3424. B, Cytotoxicity of OBI-3424 against H460 cells ± TH-3021, a specific inhibitor of AKR1C3. Cytotoxicity of OBI-3424 against a panel of six B-ALL (C), seven T-ALL (D), and six ETP-ALL (E) PDXs. Data in CE are displayed as the mean ± SEM of biological triplicates.

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To assess the potential antileukemic activity of OBI-3424, in vitro cytotoxicity assays were carried out on a broad range of leukemia cell lines. OBI-3424 exhibited potent cytotoxicity, in particular against cell lines derived from T-ALL with high AKR1C3 expression, with IC50 values in the low nmol/L range (Supplementary Table S1). The difference in IC50 values between cell lines with high/medium AKR1C3 expression and those with low expression was statistically significant (P = 0.0016). In vitro cytotoxicity assays were then carried out against a panel of 19 PDXs representative of B-ALL, T-ALL, and early T-cell precursor ALL (ETP-ALL; Supplementary Table S2). Similar to the results obtained with leukemia cell lines, OBI-3424 exerted potent cell killing against ALL PDXs (Fig. 1C–E). The median IC50 values were 60.3 nmol/L (range 3.2 nmol/L–>10 μmol/L) for B-ALL (Fig. 1C; Supplementary Table S2), 9.7 nmol/L (1.1–745 nmol/L) for T-ALL (Fig. 1D; Supplementary Table S2), and 31.5 nmol/L (4.0–130 nmol/L) for ETP-ALL (Fig. 1E; Supplementary Table S2). When cell survival relative to vehicle-treated controls was compared at 100 nmol/L OBI-3424, T-ALL PDXs were more sensitive than B-ALL and ETP-ALL PDXs (Supplementary Fig. S1).

AKR1C3 expression in pediatric ALL biopsy specimens and PDXs

We next assessed AKR1C3 expression in diverse pediatric ALL subtypes with respect to OBI-3424 sensitivity. AKR1C3 expression was significantly higher in diagnosis bone marrow aspirates from pediatric patients with T-ALL (n = 264) compared with B-ALL (n = 1,740; P < 0.0001; Fig. 2A). AKR1C3 expression in B-ALL was relatively low, especially in cases harboring DUX4 and MEF2D rearrangements (Supplementary Fig. S2A). In contrast, AKR1C3 expression in T-ALL was generally high, with the exception of subtypes harboring TLX1/3 rearrangements (Supplementary Fig. S2B). AKR1C3 expression did not differ significantly between ETP-ALL and T-ALL (Supplementary Fig. S2C). There was no evidence of genomic alterations or mutations of AKR1C3 as potential factors influencing differential expression levels of AKR1C3 between B- and T-ALL.

Figure 2.

AKR1C3 expression in primary ALL and PDX cells and quantification of DNA ICLs. RNA-seq data of primary (A) and PDX (B) B-ALL versus T-ALL cells expressed as FPKM on a log2 scale. AKR1C3 mRNA (C) and protein (D) expression levels in a panel of six B-ALL, six T-ALL, and six ETP-ALL PDXs. A representative immunoblot is shown in D. Correlations of PDX cell survival following treatment with OBI-3424 and AKR1C3 protein expression at 100 nmol/L (E) or 10 nmol/L (F) OBI-3424. G, Comet assay results showing DNA ICL indices in three ALL PDXs (ALL-8, ETP-2, and ALL-19) following OBI-3424 treatment (4 hours). H, Representative comet images from G are shown for ALL-8. In A and B, bars represent the median and interquartile range. Data in C, D, and G are displayed as the mean ± SEM of biological triplicates. Solid and dashed lines in E and F represent linear regression and 95% confidence interval, respectively.

Figure 2.

AKR1C3 expression in primary ALL and PDX cells and quantification of DNA ICLs. RNA-seq data of primary (A) and PDX (B) B-ALL versus T-ALL cells expressed as FPKM on a log2 scale. AKR1C3 mRNA (C) and protein (D) expression levels in a panel of six B-ALL, six T-ALL, and six ETP-ALL PDXs. A representative immunoblot is shown in D. Correlations of PDX cell survival following treatment with OBI-3424 and AKR1C3 protein expression at 100 nmol/L (E) or 10 nmol/L (F) OBI-3424. G, Comet assay results showing DNA ICL indices in three ALL PDXs (ALL-8, ETP-2, and ALL-19) following OBI-3424 treatment (4 hours). H, Representative comet images from G are shown for ALL-8. In A and B, bars represent the median and interquartile range. Data in C, D, and G are displayed as the mean ± SEM of biological triplicates. Solid and dashed lines in E and F represent linear regression and 95% confidence interval, respectively.

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Analysis of RNA-seq data from 90 ALL PDXs, including 25 derived from patients at relapse, confirmed significantly higher AKR1C3 expression in T-ALL (n = 25) versus B-ALL (n = 65; P < 0.0001; Fig. 2B; Supplementary Fig. S2D), and was confirmed by qRT-PCR (Fig. 2C) and immunoblotting (Fig. 2D). AKR1C3 expression was also significantly higher in T-ALL than most normal tissues (Supplementary Fig. S3), with the exception of several tissues with comparable or higher expression (adipose tissue, colon, kidney, and liver). There was a significant correlation between AKR1C3 mRNA and protein expression (R = 0.58; P = 0.0003; Supplementary Fig. S4). Moreover, AKR1C3 protein expression showed significant inverse correlations with the in vitro cell survival of the 18 ALL PDXs at OBI-3424 concentrations of both 100 nmol/L (Fig. 2E) and 10 nmol/L (Fig. 2F). Of note, an AKR1C3high B-ALL PDX (ALL-7) exhibited relative sensitivity to OBI-3424, whereas an AKR1C3low T-ALL PDX (ALL-42) was OBI-3424 resistant, highlighting the importance of AKR1C3 expression, rather than ALL lineage, in sensitivity to OBI-3424 (Supplementary Table S2).

OBI-3424 also induced a concentration-dependent increase in the ICL index in ALL-8 and ETP-2, but not ALL-19 (AKR1C3low, Fig. 2C and D; Supplementary Table S2), as assessed by comet assay (Fig. 2G and H). These results, along with the relative resistance of ALL-19 to the cytotoxic effects of OBI-3424 (Fig. 1C) are consistent with AKR1C3-dependent activation of OBI-3424 into a DNA alkylator resulting in cytotoxic DNA ICLs. The higher AKR1C3 expression in primary T-ALL cells and PDXs compared with B-ALL suggests that T-ALL may be particularly susceptible to targeting in vivo with OBI-3424.

OBI-3424 stability, preclinical pharmacokinetics, and toxicology

Because there is no functional murine equivalent of AKR1C3 (33), mouse liver cytosolic fractions are unable to activate OBI-3424. In contrast, monkey and human liver cytosolic fractions rapidly activate OBI-3424 in an AKR1C3-dependent fashion (Supplementary Fig. S5). Therefore, prior to evaluation of OBI-3424 in immunodeficient murine preclinical experimental models it was important to assess the stability, pharmacokinetics, and toxicity of OBI-3424 in relevant preclinical model systems.

OBI-3424 retained >90% stability in mouse, monkey, and human plasma for at least 2 hours (Fig. 3A), although it was most unstable in hepatic microsomes from monkeys and more stable in those from mice and humans (Fig. 3B). OBI-3424 was well tolerated in mice at doses >10 mg/kg, and OBI-3424 plasma pharmacokinetics in female H460 tumor-bearing mice (Nu-Foxn 1nu NU/NU, 5 mg/kg) and cynomolgus monkeys (2 mg/kg) are shown in Supplementary Tables S5 and S6, respectively. In a non-GLP toxicology study in cynomolgus monkeys, OBI-3424 was administered by 30 minutes i.v. infusion every 7 days × 2 for two cycles with a 1 week break between cycles. No toxicity was observed at 0.32 mg/kg, with mean white blood cell count (WBC) remaining within reference intervals for the duration of the study (Supplementary Fig. S6), whereas all animals receiving 1 mg/kg OBI-3424 were humanely killed on days 10, 12, or 13 due to severe diarrhea. Microscopic examination showed correlative severe pathology in the small intestine with villous atrophy, crypt loss, and reactive hyperplasia with necrotic cell debris.

Figure 3.

In vitro stability of OBI-3424. A, Stability of OBI-3424 in plasma. B, Stability of OBI-3424 in liver microsomes. OBI-3424 was assayed by LC/MS-MS. Data represent the mean ± relative SD (RSD). Data in A and B are displayed as the mean ± RSD (coefficient of variation).

Figure 3.

In vitro stability of OBI-3424. A, Stability of OBI-3424 in plasma. B, Stability of OBI-3424 in liver microsomes. OBI-3424 was assayed by LC/MS-MS. Data represent the mean ± relative SD (RSD). Data in A and B are displayed as the mean ± RSD (coefficient of variation).

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No OBI-3424–related clinical signs were observed in animals administered 0.32 mg/kg OBI-3424, with no marked changes in body weight, hematology, clinical chemistry, or urinalysis parameters (Supplementary Tables S7–S12 and data not shown). OBI-3424–related clinical chemistry changes were limited to increases in creatine kinase. On days 8 (13.6-fold; female 2101) and 10 (10.9- fold; female 2102 at euthanasia) in animals treated with 1 mg/kg OBI-3424, creatine kinase values increased relative to day 1 baseline values. A similar elevation was observed (15.3-fold baseline) on day 43 in 1 female (1101) treated with 0.32 mg/kg OBI-3424, and a minor increase (3.8-fold baseline) in the same animal at day 8. Therefore, OBI-3424 was well tolerated in cynomolgus monkeys at the level of 0.32 mg/kg/dose.

Extrapolation of the AUC values between mouse and monkey in Supplementary Tables S5 and S6 indicates that a dose of 0.32 mg/kg i.v. in the cynomolgus monkey approximates to 2.5 mg/kg i.p. in the mouse in terms of equivalent plasma drug exposure levels. Although this dose is well below the mouse maximum tolerated dose (MTD), the dose of 2.5 mg/kg administered every 7 days i.p. was selected as the maximum dose for all subsequent experiments to assess the in vivo efficacy of OBI-3424 against ALL PDX models in immunodeficient mice.

In vivo efficacy of OBI-3424 against PDX models of pediatric ALL

The in vivo efficacy of OBI-3424 was evaluated against seven pediatric ALL PDXs (six × T-ALL, one × B-ALL; Table 1), as well as a B-ALL PDX (ALL-11) that had previously been lentivirally transduced to overexpress AKR1C3 (ALL-11/AKR1C3) or empty vector control (ALL-11/EV; ref. 14). OBI-3424 administered as a single agent every 7 days for only three doses significantly delayed the progression of all PDXs tested by between 17.1 and 77.8 days, including those derived from patients with T-ALL who experienced fatal disease (Fig. 4; Table 1; Supplementary Fig. S7; Supplementary Table S13). Moreover, the EFS T/C values were 3.9–14.0 for T-ALL PDXs and 2.5 for the two B-ALL PDXs (ALL-28 and ALL-11/EV). The T/C value (3.5) for ALL-11/AKR1C3 was more consistent with the T-ALL PDXs, confirming the importance of AKR1C3 in the in vivo sensitivity of ALL PDXs to OBI-3424. OBI-3424 also induced regressions in eight of nine PDXs tested, with two PDXs achieving complete responses (CR) and six maintained CRs (MCR) (Table 1; Supplementary Table S13). Of note, only two of 72 mice treated with OBI-3424 were euthanized for toxicity due to reaching the predefined endpoint of ≥20% weight loss (Supplementary Table S13).

Table 1.

Results of in vivo OBI-3424 efficacy testing against a panel of nine ALL PDXs

Results of in vivo OBI-3424 efficacy testing against a panel of nine ALL PDXs
Results of in vivo OBI-3424 efficacy testing against a panel of nine ALL PDXs
Figure 4.

In vivo efficacy of OBI-3424 against ALL PDXs. Mice engrafted with ALL-8 (A), ALL-29 (B), ALL-30 (C), ALL-31 (D), ALL-11/AKR1C3 (E), and ALL-11/EV (F) were treated with OBI-3424 (2.5 mg/kg i.p. once weekly × 3 weeks, red lines) or vehicle control (black lines). The lighter hued lines represent individual mice for each treatment (vehicle or OBI-3424), whereas the darker line represents the median values for each group. Engraftment of each PDX, showing the %huCD45+ over time (left). Mouse EFS (see Table 1 and Supplementary Table S13 for P; middle). Arrows on the x axes represent the three treatments. Percent infiltration of femoral bone marrow pretreatment (gray circles), control mice at event (black squares), or OBI-3424–treated mice at day 28 posttreatment initiation (red triangles; right). BM, whole-bone marrow; LC, left central region; LE, left endosteal region; RC, right central region; RE, right endosteal region (*, P < 0.0001 comparing OBI-3424–treated and control mice at event).

Figure 4.

In vivo efficacy of OBI-3424 against ALL PDXs. Mice engrafted with ALL-8 (A), ALL-29 (B), ALL-30 (C), ALL-31 (D), ALL-11/AKR1C3 (E), and ALL-11/EV (F) were treated with OBI-3424 (2.5 mg/kg i.p. once weekly × 3 weeks, red lines) or vehicle control (black lines). The lighter hued lines represent individual mice for each treatment (vehicle or OBI-3424), whereas the darker line represents the median values for each group. Engraftment of each PDX, showing the %huCD45+ over time (left). Mouse EFS (see Table 1 and Supplementary Table S13 for P; middle). Arrows on the x axes represent the three treatments. Percent infiltration of femoral bone marrow pretreatment (gray circles), control mice at event (black squares), or OBI-3424–treated mice at day 28 posttreatment initiation (red triangles; right). BM, whole-bone marrow; LC, left central region; LE, left endosteal region; RC, right central region; RE, right endosteal region (*, P < 0.0001 comparing OBI-3424–treated and control mice at event).

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Although OBI-3424-induced substantial and prolonged regressions of disease as assessed by the surrogate marker of leukemic blasts in the peripheral blood (Fig. 4, left and middle; Supplementary Fig. S7), of particular note was the observation that OBI-3424 caused profound reductions in bone marrow infiltration of the disease at day 28 (14 days after the last OBI-3424 treatment) in six of nine ALL PDXs (Fig. 4, right; Table 1; Supplementary Fig. S7). Specifically, OBI-3424 caused significant reductions in bone marrow infiltration to <5% human versus mouse CD45+ cells in all bone marrow regions analyzed for six PDXs (ALL-8, -28, -29, -30, -31, and ALL-11/AKR1C3). Notable exceptions were ALL-27 (in which most mice were lost due to mouse-related lymphoma), ALL-32 (a PDX derived from a patient with T-ALL at relapse harboring a NUP214-ABL1 translocation; Supplementary Table S3), and ALL-11/EV (a B-ALL PDX).

We next assessed the in vivo efficacy OBI-3424 against three clinically relevant criteria: (i) the possible development of drug resistance; (ii) efficacy across a broad dose range; and (iii) efficacy in combination with standard-of-care-drugs. First, mice engrafted with all nine PDXs were subjected to retreatment with OBI-3424 after each mouse had reached event (>25% huCD45+ in the PB). Despite the repeat course of treatment occurring at a much higher disease burden than the initial treatments, seven of nine PDXs achieved objective responses, with median %huCD45+ in the PB <1% for at least 1 week (Supplementary Fig. S8; Supplementary Table S14). Second, two T-ALL PDXs derived from patients who experienced aggressive and fatal disease (ALL-8 and -31) were tested over a 5-fold reduction in OBI-3424 dose (0.5, 1.0, and 2.5 mg/kg). OBI-3424 at all three doses significantly delayed the progression of both PDXs (Fig. 5A and B, left and middle, Supplementary Table S13) and induced objective responses in ALL-31, whereas objective responses in ALL-8 were elicited at the two highest doses (1.0 and 2.5 mg/kg). The responses of both PDXs at the highest dose of OBI-3424 were remarkably similar between separate experiments (compare Fig. 5A with 4A, and 5B with 4D). Moreover, significant reductions in blood (cardiac puncture), spleen, and bone marrow infiltration were observed at day 28 for ALL-31 at all three dose levels (Fig. 5B, right), and in ALL-8 at the two highest doses (Fig. 5A, right).

Figure 5.

In vivo dose response of OBI-3424 and in combination with nelarabine. ALL-8 (A) and ALL-31 (B) dose response in mice treated with vehicle control (black lines) or OBI-3424 (0.5 mg/kg, gray lines; 1.0 mg/kg, orange lines; and 2.5 mg/kg, red lines). ALL-8 (C) and ALL-31 (D) OBI-3424 in combination with nelarabine in mice treated with vehicle control (black lines), nelarabine alone (blue lines), OBI-3424 alone (red lines), or OBI-3424 plus nelarabine (green lines). A–D, Left panels show engraftment of each PDX as the %huCD45+ over time, whereas middle panels show the mouse EFS (see Table 1 and Supplementary Table S13 for P). The lighter hued lines in the left panels represent individual mice, whereas the darker lines represent the median values for each group. Arrows indicate OBI-3424 treatment times, whereas vertical lines indicate nelarabine treatments. Right panels show the %huCD45+ in blood (cardiac puncture, CP), spleen (SPL), and femoral bone marrow pretreatment (black circles), control mice at event (open circles), and day 28 posttreatment initiation in mice treated with OBI-3424 at 0.5 mg/kg (gray squares), 1.0 mg/kg (orange triangles), 2.5 mg/kg (red squares or triangles), nelarabine (blue triangles), and OBI-3424 plus nelarabine (green triangles). BM L, left bone marrow; BM R, right bone marrow (*, P < 0.05; **, P < 0.01; ***, P < 0.0001; ****, P < 0.0001 comparing OBI-3424–treated or OBI-3424 plus nelarabine–treated and control mice at event).

Figure 5.

In vivo dose response of OBI-3424 and in combination with nelarabine. ALL-8 (A) and ALL-31 (B) dose response in mice treated with vehicle control (black lines) or OBI-3424 (0.5 mg/kg, gray lines; 1.0 mg/kg, orange lines; and 2.5 mg/kg, red lines). ALL-8 (C) and ALL-31 (D) OBI-3424 in combination with nelarabine in mice treated with vehicle control (black lines), nelarabine alone (blue lines), OBI-3424 alone (red lines), or OBI-3424 plus nelarabine (green lines). A–D, Left panels show engraftment of each PDX as the %huCD45+ over time, whereas middle panels show the mouse EFS (see Table 1 and Supplementary Table S13 for P). The lighter hued lines in the left panels represent individual mice, whereas the darker lines represent the median values for each group. Arrows indicate OBI-3424 treatment times, whereas vertical lines indicate nelarabine treatments. Right panels show the %huCD45+ in blood (cardiac puncture, CP), spleen (SPL), and femoral bone marrow pretreatment (black circles), control mice at event (open circles), and day 28 posttreatment initiation in mice treated with OBI-3424 at 0.5 mg/kg (gray squares), 1.0 mg/kg (orange triangles), 2.5 mg/kg (red squares or triangles), nelarabine (blue triangles), and OBI-3424 plus nelarabine (green triangles). BM L, left bone marrow; BM R, right bone marrow (*, P < 0.05; **, P < 0.01; ***, P < 0.0001; ****, P < 0.0001 comparing OBI-3424–treated or OBI-3424 plus nelarabine–treated and control mice at event).

Close modal

Third, we tested the in vivo efficacy of OBI-3424 in combination with the nucleoside analog nelarabine (34). At its MTD nelarabine significantly delayed the progression of ALL-8 and -31 (Fig. 5C and D, left and middle; Supplementary Table S13), but did not significantly decrease organ infiltration (Fig. 5C and D, right). The single-agent efficacy of OBI-3424 was consistent with that reported above, and OBI-3424 combined with nelarabine further delayed the progression of ALL-8 (by >156 days) and ALL-31 (by 14 days) compared with OBI-3424 alone, and compared with nelarabine alone (ALL-8, >218 days; ALL-31, 75 days; Fig. 5C and D, left and middle; Supplementary Table S13). The OBI-3424/nelarabine combination also profoundly decreased organ infiltration of both PDXs at day 28 compared with nelarabine alone or vehicle control (Fig. 5C and D, right). However, because pharmacokinetic analysis was not carried out, it was not possible to exclude the possibility that the enhanced in vivo effects of the OBI-3424/nelarabine combination were due to drug–drug interactions increasing the exposure to one or both of the drugs.

OBI-3424 is a novel prodrug that selectively releases a bis-functional DNA-alkylating agent upon reduction by AKR1C3 in the presence of NADPH. The precise nature of the DNA cross-links induced by OBI-3424 are unknown, but are likely to be similar to other ethylene imine–based DNA-alkylating drugs such as thioTEPA, which can alkylate the N-7 position of guanosine and the N-3 or N-7 position of adenosine to form monofunctional and bifunctional DNA alkylations leading to DNA strand breaks, ICLs, and intrastrand cross-links (32). Release of the aziridine moiety may also result in the formation of stable adducts with guanosine. The potent induction of DNA ICLs by low nmol/L concentrations of OBI-3424 in 2 AKR1C3high PDXs but not in an AKR1C3low PDX is consistent with AKR1C3-dependent DNA cross-linking being the major mode of cytotoxicity induced by OBI-3424 in T-ALL cells.

Although AKR1C3 is known to be overexpressed in several adult malignancies (35, 36), in particular liver (13) and prostate (37, 38) cancer, its relatively high expression in pediatric T-ALL indicates that OBI-3424 also represents a targeted therapy for T-ALL. Although the contribution of cyclophosphamide to improving patient outcomes in pediatric ALL cannot be understated, the long-term effects of treatment with this DNA-alkylating agent include reproductive problems, infertility, and secondary neoplasms. Both cyclophosphamide and OBI-3424 are prodrugs, although the former is activated by hepatic enzymes with reactive metabolites disseminating into the general circulation. In contrast, OBI-3424 is activated intracellularly to produce a potent DNA-alkylating compound, OBI-2660, which exists as a polar salt at pH 7.4 and cannot penetrate the plasma membrane, and this property has the potential to result in reduced systemic and/or bystander toxicity.

In this study using a cohort of >2,000 primary pediatric ALL cases, AKR1C3 mRNA expression was significantly higher in T-ALL compared with B-ALL, a difference that was confirmed in a separate cohort of 90 pediatric ALL PDXs. The mechanistic basis for differential regulation of AKR1C3 expression between B-ALL and T-ALL is currently unknown, but may involve additional genes regulated by the Keap1-Nrf2-Antioxidant Response Element signaling pathway including AKR1C1, AKR1C2, AKR1C4, and NQO1 (13).

OBI-3424 exerted potent in vitro cytotoxicity against T-ALL cell lines and in vitro–cultured T-ALL PDXs, with IC50 values generally in the low nmol/L range. When tested on the same cohort of pediatric T-ALL PDXs this degree of in vitro potency was approximately 1,000-fold greater than the hypoxia-activated DNA-alkylating pre-prodrug PR-104, which is also activated under aerobic conditions by AKR1C3 (13, 14). Moreover, the level of AKR1C3 expression appeared to be a more important determinant of in vitro OBI-3424 sensitivity than cell lineage, because the B-ALL PDX ALL-7 (AKR1C3high) and the T-ALL PDX ALL-42 (AKR1C3low) exhibited relative sensitivity and resistance, respectively, to OBI-3424. This inference was further supported by the significant correlation between AKR1C3 protein expression and OBI-3424 sensitivity exhibited by pediatric ALL PDXs.

When tested as a single agent in vivo OBI-3424 exerted potent efficacy, not only in the peripheral blood, but also in dramatically reducing bone marrow infiltration of T-ALL PDXs that were for the most part derived from aggressive disease. In some instances, leukemic infiltration of the femoral bone marrow was reduced to almost undetectable levels, even at 14 days following the cessation of OBI-3424 treatment. This level of single-agent efficacy compares highly favorably alongside >50 novel agents previously tested by the Pediatric Preclinical Testing Program (PPTP; ref. 39). The importance of AKR1C3 in the in vivo response to OBI-3424 was also reinforced by utilizing a B-ALL PDX (ALL-11) that had been lentivirally transduced to stably express AKR1C3.

In contrast to the vast majority of targeted single agents initially tested by the PPTP (39), OBI-3424 was initially tested at a dose well below its MTD. Because mice lack a functional equivalent of human AKR1C3 (33), the dose of OBI-3424 selected for our in vivo efficacy studies was extrapolated from pharmacokinetic data derived from mice and cynomolgus monkeys to mimic a dose that will be well tolerated in humans. The selected dose for NSG mice (2.5 mg/kg) was well tolerated by all treated mice, and was 220-fold less than the initial (MTD) dose used in the preclinical in vivo efficacy testing of PR-104 against ALL PDXs (40), with both drugs being administered weekly.

Because the development of drug resistance remains a significant barrier to cancer cure, we also tested the OBI-3424 responses of previously treated ALL PDXs following relapse of the disease. While the second cycle of treatment occurred at a much higher leukemic burden than the initial treatment, OBI-3424 was able to induce remissions in the retreated PDXs, suggesting that acute drug resistance was not induced by the original schedule of OBI-3424 treatment. Furthermore, the combination of OBI-3424 and nelarabine was also well tolerated in NSG mice and induced disease regression in two T-ALL PDXs that were derived from aggressive and fatal disease. Because nelarabine is FDA approved for relapsed T-ALL (34), the combination of OBI-3424 and nelarabine could also be considered for future clinical evaluation in relapsed/refractory T-ALL. Should OBI-3424 show activity in the relapsed/refractory setting, it may be an attractive candidate for evaluation in newly diagnosed patients (e.g., by substituting it for cyclophosphamide or by adding it to nelarabine treatment blocks).

The OBI-3424 in vivo preclinical data presented in this study should only be used to inform of potential efficacy, and both its safety and efficacy are currently being evaluated in a phase I/II clinical trial for patients with hepatocellular carcinoma or castrate-resistant prostate cancer (ClinicalTrials.gov NCT03592264). Nevertheless, the cross-species pharmacokinetic comparisons (efficacy in mice, toxicity in nonhuman primates, and allometric extrapolation to humans) suggest that there will indeed be a therapeutic window. This premise is also supported by the vastly different preclinical toxicity profile of OBI-3424 (gastrointestinal, with no significant effects on WBC) compared with the clinical DLT of myelosuppression for both cyclophosphamide and PR-104. Given the potent in vivo efficacy of OBI-3424 against preclinical models of pediatric T-ALL, its efficacy over a broad dose range, and the excellent correlation between AKR1C3 mRNA and protein expression levels, OBI-3424 represents a promising candidate for a biomarker-driven clinical trial in relapsed/refractory T-ALL.

C. G. Mulligan is an employee of Amgen and Pfizer; reports receiving commercial research grants from Loxo Oncology, Pfizer, and AbbVie; and reports receiving speakers bureau honoraria from Amgen and Pfizer. No potential conflicts of interest were disclosed by the other authors.

Conception and design: K. Evans, J. Duan, T. Pearce, M.A. Smith, R.B. Lock

Development of methodology: C.D. Jones, L. McDermott, R.B. Lock

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Evans, T. Pritchard, C.D. Jones, L. McDermott, Z. Gu, N. El-Zein, F. Meng, K.G. Roberts, C.G. Mullighan

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Evans, J. Duan, T. Pritchard, C.D. Jones, L. McDermott, Z. Gu, C.E. Toscan, N. El-Zein, C. Mayoh, S.W. Erickson, Y. Guo, F. Meng, K.S. Rathi, C.G. Mullighan

Writing, review, and/or revision of the manuscript: K. Evans, T. Pritchard, C.D. Jones, L. McDermott, S.W. Erickson, C.G. Mullighan, C.-S. Shia, T. Pearce, B.A. Teicher, M.A. Smith, R.B. Lock

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Evans, C.D. Jones, C.E. Toscan, Y. Guo

Study supervision: B.A. Teicher, R.B. Lock

Other (generated and analyzed the data): D. Jung

The authors thank Ms. Sara Danielli for assistance in setting up the comet assay. The X-RAD 320 Biological Irradiator was provided by the Biological Resource Imaging Laboratory, Mark Wainwright Analytical Centre, UNSW Sydney (Kensington, Australia). Children's Cancer Institute Australia is affiliated with UNSW Sydney and The Sydney Children's Hospitals Network. This research was funded by grants from the NCI (CA199222, CA199000, and CA197695), the National Health and Medical Research Council of Australia (NHMRC Fellowships APP1059804 and APP1157871 to R.B. Lock), and the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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