Adipocytes Sequester and Metabolize the Chemotherapeutic Daunorubicin Xia Sheng1, Jean-Hugues Parmentier1, Jonathan Tucci1, Hua Pei2, Omar Cortez-Toledo1, Christina M

Published OnlineFirst November 8, 2017; DOI: 10.1158/1541-7786.MCR-17-0338

Metabolism Molecular Cancer Research Adipocytes Sequester and Metabolize the Chemotherapeutic Daunorubicin Xia Sheng1, Jean-Hugues Parmentier1, Jonathan Tucci1, Hua Pei2, Omar Cortez-Toledo1, Christina M. Dieli-Conwright3, Matthew J. Oberley4, Michael Neely5,6, Etan Orgel6,7, Stan G. Louie2, and Steven D. Mittelman1,6,8

Abstract

Obesity is associated with poorer outcome for many cancers. conversion of daunorubicin to daunorubicinol in vivo. Adipocytes Previously, we observed that adipocytes protect acute lympho- expressed high levels of AKR and CBR isoenzymes that deactivate blastic leukemia (ALL) cells from the anthracycline, daunorubi- anthracyclines. Indeed, adipocyte protein levels of AKR1C1, cin. In this study, it is determined whether adipocytes clear AKR1C2, and AKR1C3 are higher than all other human noncan- daunorubicin from the tumor microenvironment (TME). Intra- cerous cell types. To our knowledge, this is the first demonstration cellular daunorubicin concentrations were evaluated using fluo- that adipocytes metabolize and inactivate a therapeutic drug. rescence. Daunorubicin and its largely inactive metabolite, dau- Adipocyte-mediated daunorubicin metabolism reduces active norubicinol, were analytically measured in media, cells, and drug concentration in the TME. These results could be clinically tissues using liquid chromatography/mass spectrometry important for adipocyte-rich cancer microenvironments such as (LC/MS). Expression of daunorubicin-metabolizing enzymes, omentum, breast, and marrow. As AKR and CBR enzymes metab- aldo-keto reductases (AKR1A1, AKR1B1, AKR1C1, AKR1C2, olize several drugs, and can be expressed at higher levels in obese AKR1C3, and AKR7A2) and carbonyl reductases (CBR1, CBR3), individuals, this proof-of-principle finding has important impli- in human adipose tissue, were queried using public databases and cations across many diseases. directly measured by quantitative PCR (qPCR) and immunoblot. Adipose tissue AKR activity was measured by colorimetric assay. Implications: Adipocyte absorption and metabolism of che- Adipocytes absorbed and efficiently metabolized daunorubicin to motherapies can reduce cytotoxicity in cancer microenviron- daunorubicinol, reducing its antileukemia effect in the local ments, potentially contributing to poorer survival outcomes. microenvironment. Murine studies confirmed adipose tissue Mol Cancer Res; 15(12); 1704–13. 2017 AACR.

Introduction (2), colon (3), ovary (4), and prostate (5), no mechanisms have been proven to explain these effects. One potential con- It is well established that obesity increases the risk for cancer tributor to poor cancer outcome in obesity could be the mortality (1). While obesity has been linked with poorer inadequacy of chemotherapy dosing. Excess adiposity can lead outcome from several cancers, including that of the breast to alterations in chemotherapy pharmacokinetics. Lipophilic chemotherapies can preferentially accumulate in adipose tissue 1Diabetes and Obesity Program, Center for Endocrinology, Diabetes and Metab- (6), thus increasing the volume of distribution, and reducing olism, Children's Hospital Los Angeles, Los Angeles, California. 2School of cancer cells exposure to the chemotherapy. Practices of dose Pharmacy, University of Southern California, Los Angeles, California. 3Division capping, dosing by body surface area, and adjusting for ideal or of Biokinesiology and Physical Therapy, Herman Ostrow School of Dentistry, lean body weight could further contribute to underdosing in 4 University of Southern California, Los Angeles, California. Department of obese patients. However, few studies have systematically eval- 5 Pathology, Children's Hospital Los Angeles, Los Angeles, California. Division uated how obesity alters the disposition of chemotherapies in of Infectious Diseases, Children's Hospital Los Angeles, Los Angeles, California. 6Department of Pediatrics, Keck School of Medicine, University of Southern patients. California, Los Angeles, California. 7Children's Center for Cancer and Blood Anthracyclines such as daunorubicin and doxorubicin are Diseases, Children's Hospital Los Angeles, Los Angeles, California. 8Department important chemotherapy agents used in a wide variety of cancers of Physiology and Biophysics, Keck School of Medicine, University of Southern in children and adults. We have shown that adipocytes protect California, Los Angeles, California. acute lymphoblastic leukemia (ALL) cells from a variety of che- Note: Supplementary data for this article are available at Molecular Cancer motherapies, including daunorubicin (7, 8). We report herein Research Online (http://mcr.aacrjournals.org/). that adipocytes sequester daunorubicin and metabolize it to an Current address for X. Sheng: Aptose Biosciences, San Diego, California; and inactive form, showing for the ﬁrst time that adipose tissue is a current address for S.D. Mittelman: UCLA Mattel Children's Hospital, Los drug-metabolizing organ. Angeles, California. Corresponding Author: Steven D. Mittelman, UCLA Mattel Children's Hospital, Materials and Methods 10833 Le Conte Ave., MDCC 22-315, Los Angeles, CA 90095. Phone: 323-865- Materials 6496; Fax: 310-206-5843; E-mail: [email protected] Daunorubicin and doxorubicin were purchased from Sigma doi: 10.1158/1541-7786.MCR-17-0338 Chemical Company. Daunorubicinol and doxorubicinol 2017 American Association for Cancer Research. were purchased from Santa Cruz Biotechnology. Media and

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supplements were obtained from Gibco, Thermo Fisher Scientific. Human samples FBS was purchased from Denville Scientific. All human samples were obtained and used after Institutional review board (IRB) approval and written informed consent and in Tissue culture accord with assurances filed with and approved by the U.S. þ Human ALL cell lines BV173 (Ph pre-B-ALL) and SEM (t4;11 Department of Health and Human Services. Subcutaneous pre-B-ALL) were acquired from ATCC, authenticated by short abdominal adipose tissue biopsies were collected from a subset tandem repeats by the University of Arizona Genetics core in of obese adult female postmenopausal breast cancer survivors November 2016, and tested negative for mycoplasma. ALL cell enrolled in an exercise intervention study 0–24 weeks out from lines were cultured in RPMI1640 containing 10% FBS, 1% sodium completing chemotherapy and/or radiation (ref. 10; approved by pyruvate, 1% Glutamax, and 0.1% gentamicin ("complete the University of Southern California IRB; ClinicalTrials.gov: media"), and maintained at densities between 0.5 and 2.0 NCT01140282). Subjects were randomized to a supervised com- 6 10 cells per mL in a humidified incubator with 5% CO2. The bined aerobic and resistance exercise program over 16 weeks or murine preadipocyte cell line 3T3-L1 from ATCC was differentiated usual care, and underwent biopsy at baseline and after the into adipocytes as described previously (9) and used for experi- intervention. Biopsies were collected as described previously ments between days þ7andþ11 of differentiation. As a control, (11). Adipose tissue biopsy samples were rinsed in normal saline, undifferentiated 3T3-L1 cells were irradiated with 90 Gy to induce and then transported in saline on ice to CHLA (30 minutes), senescence and plated at confluence, referred herein as 3T3-L1 where they were immediately cut into approximately 100-mg fibroblasts. Immortalized human adipocytes (ChubS7) were also sections. Some of these were cultured in complete media for 24 differentiated and cultured as described previously. Human bone hours, and then fresh media was added with daunorubicin for marrow–derived mesenchymal stem cells (MSC) were obtained experiments. While explant weight was closely matched between from Thermo Fisher Scientific and differentiated as per manufac- experiments, adipocyte number and viability were not assessed on turer's instructions with MesenPRO medium. In coculture experi- fresh mouse or human tissues. ments, approximately 2 105 ALL cells were cultured in 0.4-mm Bone marrow biopsy specimens were obtained from children pore-size polycarbonate Transwell (Corning, Inc.) over approxi- aged 10–21 diagnosed with high-risk ALL, as described previously mately 1 105 fibroblasts or adipocytes, or no feeder layer in (12), under approval of the CHLA IRB (ClinicalTrials.gov: 24-well plates. ALL cells and adipocytes were cultured with dau- NCT01317940). Biopsies from day 29 (postinduction) were norubicin for various time intervals. ALL cell viability experiments examined. were done in 96-well plates, using 0.75–1 105 initial cells. In some experiments, BV173 cells were preloaded with daunorubicin Flow cytometry for 1 hour at 37 C, pelleted, resuspended in ice-cold PBS, and FACS analysis was done using a FACScan (BD Biosciences). plated on Transwells over either no feeder or adipocytes, and then Intracellular daunorubicin was measured using the phycoerythrin collected at designated time points over the next 4 hours for flow (PE) channel, taking advantage of the natural fluorescence of cytometry. daunorubicin. MDR-1 surface expression was quantified using an allophycocyanin (APC)-conjugated anti-human MDR-1 antibody Experimental animals from BioLegend according to manufacturer's instructions. Cells All mouse experiments were approved by the Children's Hos- stained with an APC isotype control antibody were used as a pital of Los Angeles (CHLA, Los Angeles, CA) Institutional Animal negative control. DAPI was used to distinguish live cells. For all Care and Use Committee and performed in accordance with the samples, 1–5 104 events were collected. U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals. C57BL/6J diet–induced obese and control Microscopy mice (raised, respectively, on 60 kCal% or 10 kCal% fat diet from Adipocytes were grown and differentiated on poly-D-lysine– Research Diets) were purchased from The Jackson Laboratory. coated coverslips for analysis. These coverslips could then be Obese and control male mice were used as a source of adipose placed in the bottom chambers of the Transwell cocultures. Dau- tissue explants at 4–6 months of age. Mice were anesthetized with norubicin fluorescence images were acquired with an LSM 700 ketamine and xylazine, and intracardiac perfusion performed confocal system mounted on an AxioObserver.Z1 microscope with PBS until liver clearing prior to harvesting of tissues. Adipose equipped with a 63/1.4 Plan-APOCHROMAT objective lens and tissue was rinsed with cold PBS, cut into approximately 100-mg controlled with ZEN 2009 software (Carl Zeiss Microscopy). A pieces, and washed twice with RPMI plus 10% FBS prior to culture 488-nm laser with a 560-nm long-pass filter was used for fluores- in media with daunorubicin, For in vivo pharmacokinetic distri- cence excitation and emission. Transmitted laser light was collected bution, 3 obese and 3 control 13-week-old mice were injected to form a differential interference contrast (DIC) image simulta- with 5 mg/kg daunorubicin via tail vein. Mice were anesthetized 2 neously with the fluorescence image. hours after daunorubicin injection as above, and blood samples Paraformaldehyde-fixed bone marrow samples were embedded collected via intracardiac puncture, followed by intracardiac per- with paraffin. Samples were sectioned, mounted, and subjected to fusion as above. Blood, spleen, bone marrow, subcutaneous fat, antigen retrieval with citrate buffer, pH 6.0, overnight. Endoge- and omental fat were collected for daunorubicin and daunoru- nous peroxidases were inactivated with 0.3% H2O2. Nonspecific bicinol measurements using liquid chromatography/mass spec- staining was blocked with 10% normal goat serum and 1% BSA trometry (LC/MS; see below). Plasma was separated by centrifu- before staining with polyclonal rabbit anti-human AKR1C1 (Gen- gation and white blood cells (WBC) were collected using Ficoll- eTex), AKR1C2 (Cell Signaling Technology) or AKR1C3 (OriGene Paque (GE Healthcare Life Sciences) according to manufacturer's Technologies), and detected with polymerized peroxidase– protocol. Results from control and obese mice were combined as labeled goat anti-rabbit immunoglobulin (Invitrogen; mouse they were not qualitatively or statistically different. adsorbed). The reaction was detected with 3,30-diaminobenzidine

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Figure 1. Adipocytes reduce daunorubicin (DNR) concentration in nearby ALL cells. A, Daunorubicin concentration by flow cytometry based on the natural fluorescence of daunorubicin, in BV173 and SEM leukemia cells cultured with daunorubicin in Transwells with no feeder (open circles), fibroblasts (gray triangles), or adipocytes (closed circles). Doses between 60 and 200 nmol/L daunorubicin were used to achieve 50%–80% apoptosis after 48 hours of treatment. B, MDR-1 surface expression in ALL cells cultured with daunorubicin over no feeder, fibroblasts, or adipocytes. C, BV173 preloaded with daunorubicin were plated in Transwells over no feeder (open circles) or adipocytes (closed circles; n ¼ 4). Intracellular daunorubicin was measured using flow cytometry at the indicated time points. There was no significant difference in efflux between the two conditions. D, Half-lives from curves shown in C (n ¼ 4). , P < 0.05; , P < 0.01.

(Millipore) and counterstained with Harris hematoxylin (Sigma). blocked and then probed with specific primary antibodies, fol- Images were acquired on a Leica DMI6000B Inverted Microscope lowed by horseradish peroxidase (HRP)-linked secondary anti- (40/1.25) with a Color CCD Digital Camera. bodies. Bands were detected using SuperSignal West Pico Chemi- luminescent Substrate (Thermo Scientific) and luminescence qPCR analysis recorded with ImageQuant LAS 4000 (GE Healthcare Life Adipose tissue was flash frozen and then lysed with QIAzol Sciences). (Qiagen) using TissueLyzer II according to the manufacturer's protocol (Qiagen). qPCR was performed as described previously LC/MS (Sheng and colleagues; ref. 9), with the following thermal profile: Doxorubicin (50 mLat1mg/mL) was added as an internal 10 minutes at 95C followed by 40 repeats of 95C for 15 seconds, standard (daunorubicin was the internal standard when doxoru- 60C for 1 minute, and a final dissociation stage of 95C for 15 bicin and doxorubicinol were being analyzed). The entire sample seconds, 60C for 15 seconds, and 95C for 15 seconds. See was disrupted, and protein precipitated using 900 mL of ice-cold Supplementary Table S1 for primer sequences. methanol and centrifuged at 13,000 rpm at 4 C for 5 minutes. The supernatant was isolated and evaporated to dryness. Cellular Western blots residues were reconstituted with 50-mL methanol with 0.1% Human liver tissue lysate was from Abcam (ab29889). Total formic acid, and 25 mL was injected into a Shimadzu Prominence protein was extracted from adipocytes using lysis buffer HPLC linked to a Sciex API 3000. Each of the analytes was [50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.1% SDS, 1% quantified using specific multiple reaction monitoring: Nonidet I, 1 mmol/L phenylmethylsulfonylfluoride, 1% Halt 528.50!363.3, 530.60!321.30, 544.50!361.1, 546.5!363.2 Protease Inhibitor Cocktail (Thermo Scientific) and Phosphatase and 445.63!98.60, for daunorubicin, daunorubicinol, doxoru- Inhibitor Cocktail Set II (Calbiochem)]. Adipose tissue was bicin, doxorubicinol, and mitoxantrone, respectively. ground with lysis buffer using an electric rotator with glass pestle, followed by 20 strokes in a Dounce homogenizer. Lysates were Aldo-keto reductase activity assay sonicated with Bioruptor (Diagenode) for 10 minutes on ice and Activity was measured using a colorimetric assay based on centrifuged for 15 minutes at 13,000 g at 4C. The supernatant ref. 13. Briefly, a reaction mixture containing 20 mmol/L of was retained and protein concentration was quantified by BCA 9,10-Phenanthrenequinone (PQ; Sigma) and 200 mmol/L assay (Pierce Biotechnology). Proteins were separated using SDS- bNADPH (Sigma) was added to 1 mg of purified enzyme PAGE and transferred onto a nitrocellulose membrane using the (rhAKR1C3 from R&D Systems, rhCBR1 from MyBioSource) or iBlot 2 Dry Blotting System (Life Technologies). Membranes were 5 mg lysates (mouse 3T3-L1 adipocytes, human breast adipose

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Figure 2. Adipocytes sequester daunorubicin (DNR). A, Confocal microscopy pictures of adipocytes treated with different concentrations of daunorubicin (red) for 4 hours. Scale bar, 50 mm. B, Viable BV173 ALL cells cultured for 72 hours in media in which 100 nmol/L daunorubicin had been preincubated for the indicated time over no feeder (open), ﬁbroblasts (hatched), or adipocytes (solid bars; dotted line indicates initial cell number; n ¼ 4). C, Viable BV173 ALL cells cultured for 72 hours in media that had been preincubated over no feeder (open) or adipocytes (solid bars) for 48 hours with various initial daunorubicin concentrations. P values indicate comparisons to no feeder conditions (n ¼ 3). D, Viable ALL cells after 72 hours in culture with lysates of adipocytes that had been preincubated with DNR for 48 hours. P values indicate comparisons to the 0 daunorubicin lysate condition (n ¼ 3 for 100 nmol/L, n ¼ 6 for all other doses). E and F, Media doxorubicin (E) and mitoxantrone (F) concentrations after 16 hour incubation of each drug at the indicated concentrations alone (open) or over 3T3-L1 adipocytes (closed bars; n ¼ 4–6). , P < 0.05; , P < 0.01; , P < 0.001.

tissue) in sodium phosphate buffer (0.1 mol/L, pH 6.0) in a final a publicly available, mass spectrometry–based database of the volume of 100 mL/well. Stoichiometry of the reactions was deter- human proteome. Log10-normalized protein expression of AKR/ þ mined by monitoring the decrease in NADPH/H absorbance at CBR enzymes in tissues was used to generate a heatmap using 340 nm for up to 30 minutes. Specific activity (pmol/min/mg) was Microsoft Excel. determined using blank-adjusted OD/minute slope. In some m fi experiments, indomethacin or luteolin (both 100 mol/L nal Statistical analysis concentration) were added to the enzymes or lysates 5 minutes Data are shown as mean SD. All experiments were per- prior to adding the reaction mixture. formed at least three times. For flow cytometry of intracellular daunorubicin and surface MDR-1 expression, median fluores- Search of public databases cence intensity (MFI) was reported. Two-sided, paired Student To determine whether human adipose tissue expresses enzymes t tests were used to compare differences between the experi- known to metabolize anthracyclines, we evaluated four publicly mental conditions. A P value of less than 0.05 was considered fi – available gene expression pro le datasets (14 17), which includ- statistically significant. ed Affymetrix analyses of human subcutaneous and visceral adipose tissue from children and adults. Ranks for AKR and CBR genes were for analyzed each sample, independent of tissue source Results or clinical subgroup. When more than one detection probe was Adipocytes reduce daunorubicin concentration in nearby ALL assigned to a gene, the one with the highest ranks were recorded. cells To investigate whether human adipose tissue expresses these We previously showed that adipocytes protect murine leuke- enzymes at the protein level, we searched ProteomicsDB (18), mia cells from daunorubicin in both direct contact (7), and when

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Figure 3. Adipocytes metabolize daunorubicin (DNR) into daunorubicinol (DNR-ol). A, Daunorubicin and daunorubicinol concentrations in BV173 ALL cells cultured in 100 nmol/L daunorubicin alone or in transwells over adipocytes. P values indicate comparisons with ALL alone conditions. B, Daunorubicin and daunorubicinol concentrations in the adipocytes cocultured in A. C and D, Concentration of daunorubicin (C) and daunorubicinol (D) in media cultured with no feeder cells, ﬁbroblasts, or adipocytes for various times (n ¼ 4). , P < 0.05; , P < 0.01; , P < 0.001.

separated by semipermeable membranes (8). To determine proliferated better in media that had been preincubated with whether this ALL drug resistance is a consequence of decreased adipocytes when compared with fibroblasts or no feeder cells daunorubicin concentration in leukemic cells, we cultured (Fig. 2B). This ability of adipocytes to detoxify the media was human ALL cells for 24–48 hours in Transwells over no feeder detectible even with daunorubicin concentrations as high as or 1 105 adipocytes. The presence of adipocytes significantly 1,000 nmol/L, albeit with diminished efficacy (Fig. 2C). reduced daunorubicin accumulation in ALL cells, measured by Lysates from adipocytes cultured in high daunorubicin con- fluorescence (Fig. 1A). To control for total number of cells in each centrations were toxic to BV173 cells (Fig. 2D), confirming that well, additional Transwells with ALL cells over approximately 1 adipocytes did indeed sequester daunorubicin from the media 105 3T3-L1 fibroblasts were used, and showed that fibroblasts did in sufficient quantities to allow ALL cells to resist the dauno- not affect ALL cell daunorubicin concentration. The addition of rubicin cytotoxicity. Daunorubicin did not induce any signif- adipocytes did not alter the surface expression of MDR-1 found on icant morphologic changes or cell death in adipocytes by ALL cells, suggesting that reduced intracellular daunorubicin in microscopy (not shown). ALL was not the consequence of increased cellular efflux (Fig. 1B). To determine whether adipocytes sequestered other anthra- To confirm this, BV173 cells were preloaded with 18 mmol/L cyclines and related drugs, we incubated 3T3-L1 adipocytes daunorubicin for 1 hour and plated alone or over adipocytes; the with various concentrations of doxorubicin and mitoxantrone presence of adipocytes did not alter daunorubicin efflux from ALL for 16 hours. Adipocytes decreased the concentrations of both cells (Fig. 1C and D). doxorubicin (Fig. 2E) and mitoxantrone (Fig. 2F) in the media, measured by LC/MS. Adipocytes absorb anthracyclines To test whether adipocytes sequester daunorubicin from the Adipocytes metabolize daunorubicin media, 3T3-L1 adipocytes were cultured with daunorubicin for While adipocytes detoxified very high concentrations of 4 hours. Adipocytes accumulated daunorubicin (measured by daunorubicin from the media, their lysates were not as toxic fluorescence) in a concentration-dependent manner, with sig- as one would expect based on accumulation alone. We there- nal visible primarily in the cytoplasm (Fig. 2A). To investigate fore hypothesized that adipocytes were metabolizing dauno- whether this adipocyte sequestration would reduce media rubicin. As fluorescence cannot differentiate daunorubicin from daunorubicin cytotoxicity, we preincubated media with 100 its major metabolite, daunorubicinol, we used LC/MS to quan- nmol/L daunorubicin (the EC90 dose for BV173) with no tify both compounds. When cultured in daunorubicin for feeder cells, fibroblasts, or adipocytes. ALL cells survived and 16 hours, BV173 ALL cells accumulated daunorubicin, with

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Figure 4. Intact adipose tissue metabolizes daunorubicin (DNR). A and B, Daunorubicin (open) and daunorubicinol (closed bars) concentrations in media (A)and adipose tissue (B) after 100-mg portions of intact adipose explants from mice were incubated in daunorubicin for 16 hours. C, Daunorubicin and daunorubicinol concentration in 100-mg portions of human adipose tissue biopsy material after incubation in 100 nmol/L daunorubicin for 16–24 hours. D, Intracellular and tissue daunorubicin and daunorubicinol concentrations in various tissues taken 2 hours after intravenous injection of DNR at 5 mg/kg (n ¼ 6 mice). Daunorubicinol was undetectable in spleen and bone marrow cells (N.D.). nearly undetectable concentrations of daunorubicinol (Fig. 3A). In vivo daunorubicin distribution However, when adipocytes were present, ALL cell daunorubicin Although a comprehensive pharmacokinetic experiment was concentrations were only approximately one-third as high. beyond the scope of this study, we tested whether adipose tissue Adipocytes took up daunorubicin in culture, but interest- would accumulate and metabolize daunorubicin in vivo. Two ingly accumulated higher concentrations of daunorubicinol, a hours after a daunorubicin injection, plasma daunorubicin and less cytotoxic metabolite (Fig. 3B). This suggests that adipo- daunorubicinol reached similar concentrations (Fig. 4D). While cytes can deactivate daunorubicin. This finding was affirmed daunorubicin was detectible in the spleen, bone marrow, and using human MSC-derived adipocytes, which also showed circulating WBCs, little to no daunorubicinol was detected in intracellular accumulation of daunorubicin and daunorubici- these cells. In contrast, adipose tissue accumulated both dauno- nol (not shown). Furthermore, adipocytes rapidly reduced rubicin and daunorubicinol. The ratio of daunorubicinol to media daunorubicin concentration (Fig. 3C), while increasing daunorubicin was 0.60 0.26 and 0.55 0.21 in subcutaneous media daunorubicinol concentration (Fig. 3D). Both of these and omental adipose tissue, respectively. In contrast, this ratio was effects were greater than observed in undifferentiated 3T3-L1 significantly lower in WBC (0.16 0.11, P < 0.001 vs. both fibroblasts. adipose tissues), and undetectable in spleen and marrow. These findings suggest that adipose tissue actively converts daunorubi- Daunorubicin metabolism by adipose tissue cin to daunorubicinol in vivo. To test whether intact adipose tissue would sequester and metabolize daunorubicin, we incubated mouse adipose tissue Adipocytes express daunorubicin-metabolizing enzymes explants in daunorubicin. After 16 hours, adipose tissue removed There are a number of enzymes capable of converting dauno- daunorubicin from the media, replacing much of it with released rubicin to daunorubicinol (19, 20), and so we next evaluated daunorubicinol (Fig. 4A). Adipose tissue accumulated both the whether human adipose tissue expresses these enzymes. Four cytotoxic daunorubicin and the inactivated daunorubicinol, dem- publicly available gene expression profiles (14–17) showed that onstrating that intact adipose tissue can sequester, metabolize, human adipose tissue expressed high levels of many of these and inactivate daunorubicin (Fig. 4B). This experiment was per- enzymes, including aldo-keto reductase (AKR)1A1, AKR1B1, formed with adipose tissue from various anatomic sites, and AKR1C1, AKR1C2, AKR1C3, AKR7A2, carbonyl reductase demonstrated that adipose from all depots can efficiently metab- (CBR)1, and CBR3 (Fig. 5A). AKR1B10 and AKR1C4 were not olize daunorubicin to daunorubicinol. Human subcutaneous expressed (not shown). qPCR of human adipose tissue biopsy adipose tissue biopsy specimens also accumulated and metabo- specimens confirmed high gene expression of all of these lized daunorubicin, albeit with a high degree of variability in these metabolic enzymes relative to b-actin(Fig.5B),aswellas experiments (Fig. 4C). undetectable levels of AKR1B10 and 1C4. ProteomicsDB, a

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Figure 5. Expression of anthracycline-metabolizing enzymes in human adipocytes. A, Gene expression ranks of selected AKR and CBR enzymes in human adipose tissue from four publicly available gene expression proﬁles (mean rank without SD shown for clarity). B, Gene expression of AKR and CBR enzymes in human adipose tissue biopsy specimens (see text for subject details), by RT-PCR, relative to b-actin (n ¼ 4). C, Protein expression of AKR and CBR enzymes in human tissues from ProteomicsDB (18). D, Western blots showing protein expression of AKR and CBR enzymes in human adipose tissue biopsy specimen (25 mg protein), immortalized human adipocytes (ChubS7, 25 mg protein), and liver (10 mg protein) for comparison. E, IHC of bone marrow biopsy specimens from one of three representative children with ALL at day #29, after induction chemotherapy. HRP signal can be seen in adipocyte cytoplasm as a brown color. Magniﬁcation, 40; scale bar, 25 mm.

publicly available database of human cellular proteomics (18), inhibited by the AKR1C inhibitor, indomethacin, and the CBR1 showed that adipocytes express high protein levels of these inhibitor, luteolin, although the latter did not reach statistical enzymes(Fig.5C).Ofnote,thisdatabaseshowedadipocytesto significance (Fig. 6B). have the highest protein levels of AKR1C1, AKR1C2, and AKR1C3 of all noncancerous tissues evaluated. Western blots confirmed protein expression of these AKR and CBR enzymes, Discussion with the exception of CBR3, in human subcutaneous adipose In this study, we present the novel finding that adipocytes tissue biopsies and the human adipocyte cell line, ChubS7 (Fig. sequester and metabolize the anthracycline, daunorubicin. This 5D). Furthermore, AKR1C1, 1C2, and 1C3 were also shown to is the first report, to our knowledge, showing that adipocytes can be present in the cytoplasm of bone marrow adipocytes in metabolize and inactivate a therapeutic agent. This metabolism of children being treated for ALL (Fig. 5E). daunorubicin, as well as sequestration of other chemotherapies such as doxorubicin and mitoxantrone, could reduce the concen- Adipose tissue aldo-keto reductase activity tration of active drugs in adipocyte-rich microenvironments, such To further verify that adipose tissue has AKR activity, we utilized as adipose tissue, omentum, and bone marrow. This is of partic- a colorimetric assay based on NADPH (13). Lysates of both ular importance as during leukemia treatment, bone marrow subcutaneous human adipose tissue biopsies and murine 3T3- exhibits substantial fat accumulation (21). Furthermore, cancer L1 adipocytes showed AKR activity (Fig. 6A). This AKR activity was treatment induces large increases in whole body adiposity (22),

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Daunorubicin Metabolism in Adipocytes

Figure 6. Adipocytes exhibit aldo-keto reductase activity. A, AKR activity measured by PQ reduction by colorimetric assay demonstrates activity in human adipose tissue (n ¼ 5 subjects, each measured on three separate occasions), murine 3T3-L1 cells (n ¼ 7), and recombinant enzymes (n ¼ 3–4). B, Adipose tissue aldo-keto reductase activity in the presence of two putative AKR inhibitors, indomethacin and luteolin, measured by PQ reduction (n ¼ 5). , P < 0.01, two-sided paired t test.

and obesity itself has been associated with higher adipose tissue insufficient to clearly alter the drug's plasma pharmacokinetics. expression of some of these enzymes (23, 24). Together, these Because the vast majority of anthracycline clearance from plasma changes may contribute to local reduction of cytotoxic activity of occurs in the liver and kidney, peripheral adipocyte sequestration chemotherapy, leading to emergence of drug-resistant tumor cells and metabolism would not be expected to significantly alter and risk for treatment failure. We highlight a new role of the the plasma profile. This partially explains why Thompson and adipocyte in the emergence of chemotherapy resistance in the colleagues found that neither body mass index nor body fat tumor microenvironment. were significantly correlated with doxorubicin or daunorubicin Anthracyclines are broadly used in treatment regimens for a plasma clearance in children (27, 28). In fact, adiposity might be wide variety of cancers, including leukemia, lymphoma, ovarian, associated with a decreased plasma anthracycline clearance or pancreatic, breast cancers, bone, and soft tissue cancers. This new increased AUC in adults (for example, see ref. 29). However, finding that fat can sequester and deactivate cytotoxic chemo- treatment failures and relapses in leukemia are most often therapy has wide implications and may partially explain why present in the bone marrow, where our results show that obese patients have poorer clinical response when compared increased adiposity could reduce available anthracycline levels. with their leaner counterparts. In addition, the AKR and CBR As we have previously shown that ALL cells migrate into isoenzymes are highly expressed in adipocytes and are known adiposetissueundertheinfluence of the chemokine CXCL-12 to metabolize a wide range of drugs; thus, it is possible that (30), adipose tissue itself could be an unrecognized sanctuary adipocytes could impact the efficacies of other drugs in relevant site for leukemia cells, where anthracyclines are unable to reach microenvironments. therapeutic levels. Thus, adipocyte anthracycline sequestration We noted that adipocytes were less efficient in metabolizing and metabolism may contribute to survival of local leukemia doxorubicin when compared with daunorubicin. This was an clones within the bone marrow and adipocyte, thus increasing unexpected finding as these anthracyclines differ by only one the risk of residual disease at the end of induction therapy and hydroxyl group, and they are cleared by similar isoenzymes (19). eventual relapse. Decreased doxorubicin metabolism by adipocytes may reflect In addition to reducing active daunorubicin concentrations in differing enzyme affinity between the two anthracyclines. For the leukemia microenvironment, adipocyte-mediated daunoru- example, AKR1A1 metabolizes daunorubicin but not doxorubi- bicin metabolism could contribute to its major long-term toxicity, cin (25). Both daunorubicin and doxorubicin are substrates for cardiotoxicity. While much less cytotoxic to ALL cells than the AKR1B10, AKR1C1, AKR1C3, AKR7A2, and CBR1, but these parent compound, daunorubicinol has a longer half-life in both enzymes preferentially metabolize daunorubicin when compared plasma and cardiac tissue, and has been shown to dispropor- with doxorubicin (19, 26). On the other hand, others have tionately contribute to cardiac toxicity (31). Adipocyte seques- reported that AKR expression contributes to doxorubicin resis- tration and slow release of daunorubicinol could result in an tance in breast cancer cells, even in the absence of detectible increased plasma half-life of this metabolite. This line of doxorubicinol accumulation in these cells or media (20). None- thought is supported by a pharmacokinetic study showing that theless, adipocytes accumulated daunorubicin, doxorubicin, and doxorubicinol plasma clearance was decreased in children with mitoxantrone, suggesting that adipocytes can sequester all of >30% body fat (27). Thus, this mechanism may contribute to these chemotherapies from their microenvironment, with differ- the observed link between obesity and anthracycline-related ences only in subsequent intracellular metabolism. cardiotoxicity observed in animal models (32) and childhood While the rapid uptake and efficient deactivation of daunoru- cancer survivors (33). bicin by adipocytes reduces daunorubicin concentration in the There are some limitations to be considered in the data microenvironment and in nearby leukemia cells, the mass effect is presented.Whilewehaveshownthatadipocytesmetabolize

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Sheng et al.

daunorubicin in vivo,wehavenottestedwhetherthisleadsto Disclosure of Potential Conflicts of Interest lower active daunorubicin concentration in nearby cancer cells No potential conflicts of interest were disclosed. in vivo, nor whether obesity alters ALL cell daunorubicin concentrations in vivo. In addition, although we showed that Authors' Contributions adipocytes take up daunorubicin at a much higher rate than Conception and design: X. Sheng, S.G. Louie, S.D. Mittelman ALL cells and fibroblasts, we have not characterized these Development of methodology: X. Sheng, J.-H. Parmentier, J. Tucci, O. Cortez- uptake kinetics. These are important future experiments that Toledo, S.G. Louie, S.D. Mittelman Acquisition of data (provided animals, acquired and managed patients, pro- should be done to further characterize these effects and help vided facilities, etc.): X. Sheng, J.-H. Parmentier, J. Tucci, C.M. Dieli-Conwright, fi con rm the clinical relevance of the current studies. Further- E. Orgel, S.G. Louie more, we identified the presence of several anthracycline- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, metabolizing enzymes in adipocytes, but have not identified computational analysis): X. Sheng, J.-H. Parmentier, O. Cortez-Toledo, the individual contribution that each enzyme has on dauno- M.J. Oberley, M. Neely, E. Orgel, S.G. Louie, S.D. Mittelman rubicin metabolism. This level of detail is important, given the Writing, review, and/or revision of the manuscript: X. Sheng, J. Tucci, O. Cortez- Toledo, C.M. Dieli-Conwright, M. Neely, E. Orgel, S.G. Louie, S.D. Mittelman large number of these enzymes and isoforms, and their roles in Administrative, technical, or material support (i.e., reporting or organizing metabolizing hormones, medications, and toxins. Although we data, constructing databases): X. Sheng, J.-H. Parmentier, H. Pei are not the first to show that adipocytes express some AKR and Study supervision: S.G. Louie, S.D. Mittelman CBR enzymes (18), our findings that adipocytes express these enzymes at very high levels compared with other tissues (Fig. 4) Acknowledgments and that fat cells sequester and detoxify anthracyclines are The authors thank Dr. Esteban Fernandez for assistance in the CHLA Imaging highly novel. Adipose may be an underappreciated metabolic Core, and Dr. Michael Sheard and Tsen-Yin (Jackie) Lin in the CHLA FACS Core. tissue that can influence cancer outcomes by creating a sanctuary microenvironment for ALL cells. Our use of cell culture, Grant Support mouse experiments, and human tissues strengthen the veracity This work was supported by the National Cancer Institute at the NIH fi (R01 CA201444 and CA213129; to S.D. Mittelman), The Saban Research and clinical relevance of these ndings. fi fi Institute (to S.D. Mittelman), The Of ce of the Dean of the Keck School of In conclusion, this is the rst report demonstrating that adi- Medicine (to S.D. Mittelman), a Translational Research Program Award from pocytes sequester and efficiently metabolize a pharmaceutical the Leukemia & Lymphoma Society (to S.D. Mittelman and E. Orgel), The V agent. Specifically, adipocytes metabolize daunorubicin to a less Foundation for Cancer Research (to S.D. Mittelman), and the National Center toxic metabolite, and allow nearby ALL cells to evade daunoru- for Advancing Translational Science of the NIH (UL1 TR001855 and UL1 bicin-induced cytotoxicity. This finding could help explain why TR000130; to C.M. Dieli-Conwright). The costs of publication of this article were defrayed in part by the payment of obese cancer patients are at risk of having a poorer outcome. fi page charges. This article must therefore be hereby marked advertisement in Pharmacokinetic studies speci cally in the tumor microenviron- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ment will be necessary to determine the precise impact of adiposity on anthracycline-based treatment and efficacy in patients Received June 28, 2017; revised August 10, 2017; accepted August 28, 2017; with ALL and other cancers. published OnlineFirst November 8, 2017.

References 1. Calle EE, Rodriguez C, Walker-Thurmond K, Thun MJ. Overweight, obesity, 10. Dieli-Conwright CM, Mortimer JE, Schroeder ET, Courneya K, Demark- and mortality from cancer in a prospectively studied cohort of U.S. adults. Wahnefried W, Buchanan TA, et al. Randomized controlled trial to evaluate N Engl J Med 2003;348:1625–38. the effects of combined progressive exercise on metabolic syndrome in 2. Chan DS, Vieira AR, Aune D, Bandera EV, Greenwood DC, McTiernan A, breast cancer survivors: rationale, design, and methods. BMC Cancer et al. Body mass index and survival in women with breast cancer— 2014;14:238. systematic literature review and meta-analysis of 82 follow-up studies. 11. Alderete TL, Sattler FR, Sheng X, Tucci J, Mittelman SD, Grant EG, et al. A Ann Oncol 2014;25:1901–14. novel biopsy method to increase yield of subcutaneous abdominal adipose 3. Sinicrope FA, Foster NR, Sargent DJ, O'Connell MJ, Rankin C. Obesity is an tissue. Int J Obes 2015;39:183–6. independent prognostic variable in colon cancer survivors. Clin Cancer Res 12. Orgel E, Mueske NM, Sposto R, Gilsanz V, Freyer DR, Mittelman SD. 2010;16:1884–93. Limitations of body mass index to assess body composition due to 4. Purcell SA, Elliott SA, Kroenke CH, Sawyer MB, Prado CM. Impact of body sarcopenic obesity during leukemia therapy. Leuk Lymphoma. 2016 Jan weight and body composition on ovarian cancer prognosis. Curr Oncol 27. [Epub ahead of print]. Rep 2016;18:1–11. 13. Shultz CA, Quinn AM, Park JH, Harvey RG, Bolton JL, Maser E, et al. 5. Ma J, Li H, Giovannucci E, Mucci L, Qiu W, Nguyen PL, et al. Prediagnostic Specificity of human aldo-keto reductases, NAD(P)H:quinone oxidore- body-mass index, plasma C-peptide concentration, and prostate cancer- ductase, and carbonyl reductases to redox-cycle polycyclic aromatic hydro- specific mortality in men with prostate cancer: a long-term survival anal- carbon diones and 4-hydroxyequilenin-o-quinone. Chem Res Toxicol ysis. Lancet Oncol 2008;9:1039–47. 2011;24:2153–66. 6. Hanley MJ, Abernethy DR, Greenblatt DJ. Effect of obesity on the phar- 14. Tam CS, Heilbronn LK, Henegar C, Wong M, Cowell CT, Cowley MJ, et al. macokinetics of drugs in humans. Clin Pharmacokinet 2010;49:71–87. An early inflammatory gene profile in visceral adipose tissue in children. Int 7. Behan JW, Yun JP, Proektor MP, Ehsanipour EA, Arutyunyan A, Moses AS, J Pediatr Obes 2011;6:e360–3. et al. Adipocytes impair leukemia treatment in mice. Cancer Res 15. Keller P, Gburcik V, Petrovic N, Gallagher IJ, Nedergaard J, Cannon B, 2009;69:7867–74. et al. Gene-chip studies of adipogenesis-regulated microRNAs in 8. Sheng X, Tucci J, Parmentier JH, Ji L, Behan JW, Heisterkamp N, et al. mouse primary adipocytes and human obesity. BMC Endocr Disord Adipocytes cause leukemia cell resistance to daunorubicin via oxidative 2011;11:7. stress response. Oncotarget 2016;7:73147–59. 16. Aguilera CM, Gomez-Llorente C, Tofe I, Gil-Campos M, Canete R, Gil A. 9. Sheng X, Tucci J, Malvar J, Mittelman SD. Adipocyte differentiation is Genome-wide expression in visceral adipose tissue from obese prepubertal affected by media height above the cell layer. Int J Obes 2014;38:315–20. children. Int J Mol Sci 2015;16:7723–37.

1712 Mol Cancer Res; 15(12) December 2017 Molecular Cancer Research

Daunorubicin Metabolism in Adipocytes

17. Hardy OT, Perugini RA, Nicoloro SM, Gallagher-Dorval K, Puri V, 25. Bains OS, Takahashi RH, Pfeifer TA, Grigliatti TA, Reid RE, Riggs KW. Two Straubhaar J, et al. Body mass index-independent inﬂammation in allelic variants of aldo-keto reductase 1A1 exhibit reduced in vitro metab- omental adipose tissue associated with insulin resistance in morbid olism of daunorubicin. Drug Metab Dispos 2008;36:904–10. obesity. Surg Obes Relat Dis 2011;7:60–7. 26. Bains OS, Karkling MJ, Lubieniecka JM, Grigliatti TA, Reid RE, Riggs KW. 18. Wilhelm M, Schlegl J, Hahne H, Gholami AM, Lieberenz M, Savitski MM, Naturally occurring variants of human CBR3 alter anthracycline in vitro et al. Mass-spectrometry-based draft of the human proteome. Nature metabolism. J Pharmacol Exp Ther 2010;332:755–63. 2014;509:582–7. 27. Thompson PA, Rosner GL, Matthay KK, Moore TB, Bomgaars LR, Ellis KJ, 19. Bains OS, Grigliatti TA, Reid RE, Riggs KW. Naturally occurring variants et al. Impact of body composition on pharmacokinetics of doxorubicin in of human aldo-keto reductases with reduced in vitro metabolism children: a Glaser Pediatric Research Network study. Cancer Chemother of daunorubicin and doxorubicin. J Pharmacol Exp Ther 2010;335: Pharmacol 2009;64:243–51. 533–45. 28. Thompson P, Wheeler HE, Delaney SM, Lorier R, Broeckel U, Devidas M, 20. Heibein AD, Guo B, Sprowl JA, Maclean DA, Parissenti AM. Role of aldo- et al. Pharmacokinetics and pharmacogenomics of daunorubicin in chil- keto reductases and other doxorubicin pharmacokinetic genes in doxoru- dren: a report from the Children's Oncology Group. Cancer Chemother bicin resistance, DNA binding, and subcellular localization. BMC Cancer Pharmacol 2014;74:831–8. 2012;12:381. 29. Rodvold KA, Rushing DA, Tewksbury DA. Doxorubicin clearance in the 21. Daldrup-Link HE, Henning T, Link TM. MR imaging of therapy-induced obese. J Clin Oncol 1988;6:1321–7. changes of bone marrow. Eur Radiol 2007;17:743–61. 30. Pramanik R, Sheng X, Ichihara B, Heisterkamp N, Mittelman SD. Adipose 22. Carneiro IP, Mazurak VC, Prado CM. Clinical implications of sarcopenic tissue attracts and protects acute lymphoblastic leukemia cells from che- obesity in cancer. Curr Oncol Rep 2016;18:62. motherapy. Leuk Res 2013;37:503–9. 23. Blouin K, Blanchette S, Richard C, Dupont P, Luu-The V, Tchernof A. 31. Platel D, Bonoron-Adele S, Robert J. Role of daunorubicinol in daunoru- Expression and activity of steroid aldoketoreductases 1C in omental bicin-induced cardiotoxicity as evaluated with the model of isolated adipose tissue are positive correlates of adiposity in women. Am J Physiol perfused rat heart. Pharmacol Toxicol 2001;88:250–4. Endocrinol Metab 2005;288:E398–404. 32. Mitra MS, Donthamsetty S, White B, Mehendale HM. High fat diet-fed 24. Michaud A, Lacroix-Pepin N, Pelletier M, Veilleux A, Noel S, Bouchard obese rats are highly sensitive to doxorubicin-induced cardiotoxicity. C, et al. Prostaglandin (PG) F2 alpha synthesis in human subcutaneous Toxicol Appl Pharmacol 2008;231:413–22. and omental adipose tissue: modulation by inﬂammatory cytokines 33. Lipshultz SE, Cochran TR, Franco VI, Miller TL. Treatment-related cardi- and role of the human aldose reductase AKR1B1. PLoS One 2014;9: otoxicity in survivors of childhood cancer. Nat Rev Clin Oncol 2013; e90861. 10:697–710.

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Adipocytes Sequester and Metabolize the Chemotherapeutic Daunorubicin

Xia Sheng, Jean-Hugues Parmentier, Jonathan Tucci, et al.

Mol Cancer Res 2017;15:1704-1713. Published OnlineFirst November 8, 2017.

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