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Switch to low-fat diet improves outcome of acute lymphoblastic leukemia in obese mice

Switch to low-fat diet improves outcome of acute lymphoblastic leukemia in obese mice Background: It is becoming increasingly recognized that weight and nutritional status can impact cancer survival. We have previously shown that obese mice with syngeneic acute lymphoblastic leukemia (ALL) have poorer response to chemotherapy treatment than control mice. We therefore investigated whether dietary intervention could improve outcome from the most common pediatric cancer, ALL. Methods: Diet-induced obese (DIO) mice raised on a 60% calories from fat diet and control mice were implanted with syngeneic ALL cells. Some DIO mice were switched to the low-fat control diet. Survival from ALL was assessed without or with chemotherapy treatment starting at the time of the diet switch. Cells from DIO mice before and after diet switch were assessed by FACS for BrdU incorporation and phosphorylation status of AKT, S6K, and EIF2a. Similar experiments were done with human ALL xenografts. Mouse and human ALL cells were cultured in media with 10% or 5% fetal bovine serum, and sensitivity to chemotherapies assessed. Results: DIO mice had poorer survival (17%) after vincristine monotherapy than control mice on a 10% low fat diet (42%; n = 12/group; p = 0.09, log rank). However, switching obese mice to the low-fat diet prior to initiation of vincristine led to dramatically improved survival (92%, p < 0.01 vs both other groups). In vitro, FBS restriction made murine and human ALL cells more sensitive to vincristine. Interestingly, while serum restriction enhanced ALL sensitivity to dexamethasone and L-asparaginase, dietary switch did not improve survival of DIO mice treated with either drug in monotherapy. Thus, it appears that dietary intervention has a unique effect to improve ALL cell sensitivity to vincristine in vivo. Conclusions: We report herein that a dietary intervention can improve ALL outcome in a preclinical model. Further work is needed to identify the mechanisms of this effect and investigate potential impact on human leukemia in patients. Keywords: Obesity, Adipose tissue, Dietary intervention, Chemotherapy, Caloric restriction Background (HR-ALL) by 50% [2], a finding confirmed in meta-analysis Obesity increases the incidence of many cancer types, and [3]. In a separate cohort, we reported that obese children obese cancer patients have a higher risk of mortality from were 2.74 times more likely to be minimal residual disease their disease [1]. In 2007, a retrospective review of two large (MRD) positive [4], which portends increased relapse risk cohorts demonstrated that obesity at the time of diagnosis and reduced event-free survival [5]. Moreover, recent stud- increases risk of relapse in children with National Cancer ies show 1 in 3 children with ALL were overweight or obese Institute/Rome High-Risk acute lymphoblastic leukemia at diagnosis [6, 7]. Much attention has been given to the role of diet on cancer outcomes. Meta-analysis has shown that cancer * Correspondence: smittelman@mednet.ucla.edu survivors who maintain high quality diets have lower over- Diabetes and Obesity Program, Center for Endocrinology, Diabetes and all mortality, though no significant reduction in cancer Metabolism, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA recurrence [8]. Less clear is whether dietary intervention Division of Pediatric Endocrinology, UCLA Children’s Discovery and during cancer treatment might improve outcome. Caloric Innovation Institute, David Geffen School of Medicine UCLA, 10833 Le Conte restriction has garnered much attention in this realm, as it Ave, Los Angeles, CA 90095-1752, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tucci et al. Cancer & Metabolism (2018) 6:15 Page 2 of 8 can potently reduce insulin-like growth factor 1 (IGF-1), a Mouse models stimulator of cellular metabolism through activation of the High fat diet-induced obese (DIO) and control C57Bl/6J PI3K/Akt/mTOR axis [9, 10]. Non-transformed cells mice were purchased from Jackson Laboratories (Bar respond to this low nutrient state by downregulating Harbor, MI, USA). Mice had been raised on either a 60% mTOR activity and entering a state of quiescence, which (obese) or 10% (control) calories from fat diet (Research can protect them from some chemotherapies; however, Diets, D12492 and D12450B, respectively). Because cancer cells often exhibit constitutive activation of PI3K/ female mice do not become as significantly obese in this Akt/mTOR [11]. This constitutive activation cannot be model and are not available as DIO mice from Jackson modulated by nutrient restriction, leading to retained or in- Laboratories, only male mice were used for these stud- scid tm1Wjl creased chemosensitivity of cancer cells [12, 13]. Additional ies. Male NSG mice (NOD.Cg-Prkdc Il2rg /SzJ) mechanisms also likely contribute to the beneficial effects are NOD/SCID IL2-receptor gamma chain knockout of caloric restriction, including reduced fuel availability, re- mice commonly used for xenograft. NSG mice were duction of inflammation, and lower oxidative stress [9, 14]. made obese using selective culling and a high-fat diet as We have developed a mouse model which recapitu- previously described [19]. lates the clinical observation that obese children have worse ALL outcome; high-fat diet-induced obese (DIO) ALL survival experiments mice implanted with syngeneic ALL had a poorer sur- ALL implantation experiments were performed on DIO vival outcome after treatment with either vincristine and control mice at ~ 20 weeks of age. At this time, (VCR) or L-asparaginase [15, 16]. However, whether the 10,000 GFP pre-B-cell ALL 8093 cells were implanted effects of obesity on ALL outcome in mice or patients is retro-orbitally. 6 or 7 days after ALL implantation, reversible remains unknown. While caloric restriction depending on the experiment, DIO mice were random- has been shown to improve chemotherapy efficacy in ized to continue on their high-fat diet or switched to the mouse models of solid tumors [12], it has not been control diet (10% calories from fat, provided ad libitum). tested in the most common childhood cancer, acute In some experiments, monotherapy with vincristine lymphoblastic leukemia. Therefore, we designed the (0.5 mg/kg/week intraperitoneal) was started on day 7, present study to test whether a dietary intervention and this dose was adjusted for body weight each week. could improve ALL outcome in obese mice. Similar experiments were performed with L-asparaginase (800 IU/kg/day, Monday–Friday) or dexamethasone (8 mg/kg/day, Monday–Friday). In other experiments, Methods DIO and control NSG mice were implanted with LAX7 Cell culture cells, and after a 17 day engraftment period, half of the Murine pre-B ALL cells were previously isolated from a DIO mice switched to control diet. On day 18, treatment BCR/ABL transgenic mouse (“8093 cells” [17]) and with vincristine, L-asparaginase and dexamethasone transduced with GFP. Human leukemia cell lines in- (same doses as above) was started and continued for cluded BV173 (pre B Ph + ALL, ATCC) and Nalm-6 (B 4 weeks. Mice in all survival experiments were moni- cell precursor leukemia, ATCC). Cells were authen- tored daily for food intake and body weight, and onset ticated by the University of Arizona Genetics core in of progressive leukemia (paralysis, hunched posture, November 2016 and tested negative for mycoplasma. palpable mass > 1 cm, poor grooming, etc.), at which Eight thousand ninety-three cells were cultured in time they were euthanized. McCoy’s 5A media (Invitrogen), supplemented with 1 mM sodium pyruvate, 2 mM Glutamax, 10 μg/mL Tissue harvesting and flow cytometry gentamycin and either 5% or 10% FBS (Denville Scientific In some experiments, mice were anesthetized with keta- or Omega Scientific). Human cell lines were cultured in mine/xylazine and perfused with PBS for organ harvest RPMI 1640 (Invitrogen), supplemented as above. at various timepoints before and after ALL implantation. Chemotherapy sensitivity experiments were performed When performing cell cycle analysis, mice were injected over 72 h, with surviving viable cells counted by with BrdU intraperitoneally 4 h prior to organ harvest. blinded observers in triplicate using trypan blue exclu- Spleen and femurs were removed, and spleen pulp and sion manually, or with a Countess II (ThermoFisher). femoral marrow were extruded with red blood cell lysis LAX7 cells are patient-derived ALL cells with normal buffer (BD Biosciences, San Jose, CA, USA) for flow cy- karyotype which were expanded by passage through tometry. Red blood cell-free bone marrow and spleen NSG mice (mice described below) and used for xeno- pulp were filtered through 40 μm cell strainers to create graft [18]. Cells were cultured on OP-9 stroma cells single cell suspensions. Cells utilized for cell cycle ana- with αMEM supplemented with 20% FBS, 100 IU/mL lysis were processed according to the manufacturer’s penicillin, and 100 μg/mL streptomycin. protocol (APC BrdU Flow Kit, BD Biosciences). Cells Tucci et al. Cancer & Metabolism (2018) 6:15 Page 3 of 8 not used for cell cycle analysis were fixed in 4% PFA at relative sparing of carbohydrate intake, due to the in- 37 °C for 10 min and then permeabilized in chilled creased carbohydrate density of the control diet. After a methanol for 30 min. Cells were washed and incubated couple of weeks, food intake in the switched mice in- in Mouse Fc Block (BD Biosciences) for 15 min then in- creased, and nutrient intake became similar to that of cubated for 1 h in the following fluorophore-conjugated controls. antibodies: pEIF2α(Ser51)-PE/Cy7, pAKT(Ser473)-A647, Dietary restriction in animals leads to a large number pS6K(Thr412)-PE (Bioss USA, Boston, MA, USA). Cells of hormonal, physical, physiologic, and metabolic were analyzed using the LSR II Flow Cytometer (BD changes that cannot be completely modeled in vitro. Biosciences) in the CHLA Flow Cytometry Core. Cells However, FBS restriction can simulate some of the de- were initially gated to exclude DAPI-positive dead cells cline in growth factors that are observed with caloric re- and forward and side scatter to exclude debris. Gating striction and weight loss. Reducing FBS concentration on GFP allowed analysis of ALL vs. host cells. increased VCR cytotoxicity (Fig. 2a); however, FBS con- centrations below 5% impaired cell growth and viability. We therefore used 5% FBS, which did not affect their Data analysis proliferation rate but made them significantly more sen- Kaplan Meier survival curves were generated and com- sitive to VCR (Fig. 2b, c). Similar VCR sensitization was pared using Cox regression. Food intake was measured observed in human ALL cell lines as well, BV173 and manually by mouse cage, and reported as daily averages Nalm6 (Fig. 2d–f). or three-day moving averages as described in results. Vi- able cells and proportions of cells labeled with BrdU or Diet restriction does not alter ALL response to L- other antibodies were compared between diet switch asparaginase or dexamethasone in vivo and DIO mice using two-sided, paired t-tests. EC was Similar ALL survival experiments were done as above, calculated by fitting normalized dose-response data to but using L-asparaginase or dexamethasone monother- the equation: %viable cells ¼ . All ana- ðLogðdoseÞ−LogðEC50ÞÞ 1þ10 apy starting on day 7. Diet switch had no detectible lysis was performed using GraphPad Prism and Micro- effect on survival in these experiments (Fig. 3a, b). FBS soft Excel. restriction did increase 8093 cell sensitivity to dexa- methasone (EC 9.1 ± 2.3 vs. 16.3 ± 3.7 nM, p = 0.02) Results and tended to increase sensitivity to L-asparaginase Diet restriction sensitizes ALL cells to vincristine (EC 0.80 ± 0.22 vs. 1.30 ± 0.21 IU/mL, p =0.13; Fig. 3c, d), To determine whether switching diet could improve ALL but not to Ara-C or daunorubicin (Additional file 1:Figure treatment outcome, we implanted 16 DIO and 8 S1). In addition, survival experiments performed with hu- non-obese control mice with syngeneic ALL cells (day 0; man ALL xenografted NSG mice treated with a combin- Fig. 1a). On day 7, half of the DIO mice were switched to ation of vincristine, L-asparaginase, and dexamethasone the low-fat control diet, while the rest were continued on (VDL)showedthatdiet-restricted mice hadnodifference their regular diet (final n = 8 per group). Time to progres- in survival from DIO or control mice (Additional file 2:Fig- sive ALL was not affected by diet in these mice (Fig. 1b). ureS2).Thus, theeffect of dietaryrestriction to sensitize In other groups of mice, VCR was started on day 7, and ALL to chemotherapy treatment in vivo appears to be rela- VCR dose adjusted weekly based on body weight. In this tively specific to VCR. experiment, DIO mice switched to the low-fat diet had the best survival, which was significantly better than mice Diet restriction does not alter cell cycle rate of host or maintained on the high-fat diet (p < 0.001), and even bet- ALL cells ter than control mice that had been raised on the low-fat To investigate whether dietary restriction changes cell diet (p <0.01; n = 6 repeated in two separate experiments sensitivity to VCR by altering ALL or host cell cycling for final n = 12 per group; Fig. 1c). Overall survival at the rate, cells were harvested from groups of DIO mice be- end of the experiment was 92% in the diet-switch group, fore or after diet switch, 4 hours after BrdU injections. 42% in the control group and 17% in the DIO group. ALL and host cells were separated by FACS from Diet-switched mice in additional experiments exhibited spleens and bone marrows of DIO mice on day 6 after rapid weight loss, where their body weight declined to ALL implantation (before any diet switch), day 8 (1 day match those of the control mice within approximately after diet switch), or day 14 (7 days after diet switch). 6days (Fig. 1d). This was associated with a substantial de- The percentage of GFP+ ALL cells in S phase were de- cline in food intake, despite access to ad libitum feed. termined and compared to GFP-host cells (splenocytes Switched mice reduced food intake for several days after or marrow cells) from the same tissues. The percentage the diet switch, leading to substantially decreased intake of cells in S phase was higher in splenic ALL cells than of total calories, protein and fat (Fig. 1e). There was a non-ALL cells, but similar between ALL and non-ALL Tucci et al. Cancer & Metabolism (2018) 6:15 Page 4 of 8 Fig. 1 Dietary restriction improves obese leukemic C57BL/6J mouse survival after vincristine treatment. a Scheme showing diet intervention in survival experiments. b Survival of mice with ALL in each diet group with no chemotherapy treatment (n = 12/group). c Survival of mice with ALL in each diet group treated with VCR. ▼ indicate VCR doses. Vehicle-treated mice were distributed evenly between all three diet groups, and were combined due to no difference in survival. N = 12/group; **p < 0.01, ***p < 0.001, log rank. d Body weight of mice on each diet. e Total (left), carbohydrate (center), and fat (right) calorie daily intake in mice from each diet group. Intake is shown as daily average up until day 5 after the diet switch, and as 3 point moving average thereafter, due to high day-to-day variability. Standard deviations reflect variance between 2 and 4 cages, not individual mice. Since protein content was identical between diets, the relative intake was proportional to total caloric intake and not shown separately cells from marrow (Additional file 3: Figure S3). Diet samples (Additional file 3: Figure S3), confirming the switch did not significantly alter this percentage in either higher metabolic rate in cancer cells. However, diet switch cell type. had no significant effect on phosphorylation state of any of these proteins in host or ALL cells. Diet restriction does not alter AKT signaling in host or ALL cells Discussion To determine how diet restriction might alter relevant We report herein that switching obese mice from a intracellular signaling pathways, spleen- and marrow-de- high-fat to a low-fat diet improves ALL survival with rived cells from obese and obese-dieted mice (described vincristine treatment from 17 to 92%. This is the first above) were labeled with antibodies for EIF2a/pEIF2a, study of which we are aware testing a diet intervention AKT/pAKT, and S6K/pS6K and analyzed by flow cytome- on treatment outcome from a hematological malignancy. try. ALL cells from both tissues showed higher phosphor- Other studies have evaluated whether dietary interven- ylation levels of these proteins than host cells in most tion can affect treatment outcome of solid cancers. Tucci et al. Cancer & Metabolism (2018) 6:15 Page 5 of 8 Fig. 2 Serum restriction sensitizes ALL to vincristine treatment. a Viable 8093 cells after 72 h in culture media with various concentrations of FBS, alone, or with 10 nM VCR (n = 3). b Dose response of murine ALL 8093 cells cultured with VCR in 5 or 10% FBS (n = 4). c Viable 8093 cells after culture alone or with 10 nM VCR (n = 7) ***p < 0.001, two-sided, paired t test. d EC values calculated from dose responses of vincristine with all three cell lines; connecting lines show paired experiments. *p < 0.05, ***p < 0.001, two-sided, paired t test. e, f Dose response of human ALL cell lines BV173 (e) and Nalm6 (f) with VCR in 5 or 10% FBS (n =4) Fig. 3 Diet restriction does not sensitize murine ALL cells to dexamethasone or L-asparaginase. a, b Survival of mice in each diet group treated with dexamethasone (a, n =5–6) or L-asparaginase (b, n = 6). c, d Viable cells after 72 h in culture media with 5 or 10% FBS, alone or with 70 nM dexamethasone (c) or 2.5 IU/mL L-asparaginase (d), n =3 Tucci et al. Cancer & Metabolism (2018) 6:15 Page 6 of 8 Switching DIO mice from a high-fat to low-fat diet, which would require extensive additional experiments similar to the present study, has been shown to improve and should be evaluated in future studies. survival from melanoma in mice treated with dacarba- It is also possible that the protective effect of a dietary zine [20]. Ketogenic diets, containing very low carbohy- switch is mediated in part by immunological mecha- drates, are used for patients with intractable epilepsy nisms. In the 2010 update to the well-known “Hallmarks and cause decreases in blood glucose concentrations but of Cancer,” four new principles were identified as vital to elevations in circulating free fatty acids and ketones [21]. carcinogenesis and cancer cell survival, one being eva- These diets have been shown to have an anti-tumor ef- sion of immune destruction [25]. In addition to their fect in 12 separate murine cancer models [22] and are direct cytotoxic effect, some chemotherapies have been being evaluated in patients. Fasting and short-term star- identified as inducers of immunogenic cell death, vation has also been shown to improve treatment out- whereby their mechanism of cytotoxicity invokes a host come from a wide variety of solid tumors in mice [12]. T-cell-mediated immunologic response [26]. These in- How efficacious these diets will be in cancer patients re- clude the anti-microtubule agents docetaxel and pacli- mains to be seen. taxel, as well as the anthracyclines, daunorubicin and Our findings are consistent with retrospective data doxorubicin, and the anthracendione, mitoxantrone. which shows that reversing obesity may be associated Interestingly, caloric restriction and short-term fasting with improved ALL outcome in youth. Orgel et al. ob- have been shown to improve T-cell anti-tumor reactivity served that ALL patients who had spontaneously lost and reduce immune-suppressing T-regs. Combined, weight and changed from obese to non-obese category these studies suggest that dietary restriction could syner- over their treatment had better outcome than those who gize with a certain subset of chemotherapies to augment remained obese for > 50% of their treatment course [23]. host anti-tumor response and improve treatment out- Based on this observation, and our current findings, we comes. This would be consistent with our finding that have launched a clinical intervention trial of mild dietary NSG mice, which lack immune systems, obtained no restriction and increased physical activity in children benefit from the dietary switch. On another note, it is with newly diagnosed ALL (IDEAL Weight in ALL Trial; particularly interesting that three of the other four iden- NCT 02708108). This and similar studies are critical to tified immune inducers, daunorubicin, doxorubicin, and help determine which cancers respond to dietary inter- mitoxantrone, are commonly used in primary and re- ventions and which specific interventions work for lapse ALL chemotherapy regimens. These chemothera- which cancers. peutic agents were not studied in our current model but While we were unable to identify the mechanism should be tested in future studies. whereby dietary restriction improved ALL outcome in Our study has several limitations. Perhaps our biggest our study, we did rule out some important mecha- limitation is that we did not detect an effect of dietary nisms. VCR cytotoxicity is cell cycle dependent, as op- intervention in our human xenograft model. This limits posed to L-asparaginase and dexamethasone, which the degree to which our results may be directly translat- could explain why mice treated with dexamethasone able to human leukemia. The lack of effect in this model or L-asparaginase derived no survival benefit from a could have been due to the triple chemotherapy regimen dietary switch. However, we did not detect an effect of used, which could have masked a small effect of vincris- dietary intervention on cell cycle of ALL or host cells tine alone, to the immunodeficient state as discussed one or 7 days after diet initiation. We also found no above, or to other mouse strain differences. In any case, effect of diet intervention on the ALL or host cell ac- dietary intervention in human xenograft models should tivity of pathways commonly evaluated for effects of be further evaluated so that relevance to human dietary intervention on cancer, though we did observe leukemia can be confirmed. Another limitation is that an increased activity of these pathways in the cancer we tested a relatively crude dietary intervention—switch- cells vs. host cells. Lack of effect of diet on AKT or ing DIO mice from a high-calorie/high-fat diet to a S6K phosphorylation implies that our intervention lower-calorie/low-fat diet. This switch induced several was not acting through IGF-1 or mTOR, respectively. changes, including decreased calorie, protein, and fat in- eIF2a phosphorylation is observed with amino acid take, while sparing carbohydrate intake. These changes deprivation, oxidative stress, and unfolded protein re- undoubtedly altered the animals’ insulin sensitivity, cir- sponse [24], arguing against these potential mecha- culating growth factors, body composition, and fuel nisms of our dietary intervention. It is possible that availability. Indeed, another study which utilized a simi- these signals may have mediated effects after 7 days, lar high-fat to low-fat diet switch in C57Bl/6 mice which we would have missed with our experimental showed a number of physiological effects, including design. Further, while these findings argue against decreases in glucose and insulin, improvement in lipids these pathways, we did not exhaustively rule them out, and free fatty acids, and decrease in several pro- Tucci et al. Cancer & Metabolism (2018) 6:15 Page 7 of 8 inflammatory cytokines and adipokines [20]. This broad Acknowledgements The authors would like to thank Ehsan Ehsanipour for assistance in range of effects may contribute to the efficacy of this performing these studies. intervention, but at the same time makes identification of specific mechanisms difficult. Thus, further study will Funding This work was supported by R01CA201444 (SDM), R01CA213129 (SDM), need to be done to determine which of these changes R01CA172896 (YMK), and funds from The Saban Research Institute, CHLA. and effects contribute to the improved survival. On the other hand, murine obesity induced by a high-fat diet is Availability of data and materials The datasets used and/or analyzed during the current study are available not completely analogous to human obesity, which is from the corresponding author on reasonable request. generally associated with excess carbohydrate intake; thus, utility of these models for this purpose may be lim- Authors’ contributions JT, WA, and TC designed and performed experiments, analyzed and ited. Finally, despite the large observed effect of dietary interpreted data, and helped in the preparation of the manuscript. XS helped intervention on vincristine efficacy, the mechanism be- perform some experiments and develop flow cytometry experiments. YMK hind this effect remains elusive. provided patient sample cells and advised on xenograft model experiments. SDM conceived of the experiments, oversaw all aspects of the study, and While treatment of childhood ALL has led to substantial wrote the manuscript. All authors approved the final manuscript. improvements in survival, there are still ~ 8% children who relapse from this disease every year [27]. In addition, Ethics approval All studies were approved by the CHLA IACUC and performed in accordance children with certain ALL subtypes, adolescents and with the US Public Health Service Policy on Humane Care and Use of adults, and obese patients are all at increased risk of Laboratory Animals. relapse and mortality. Given the substantial toxicity of Consent for publication current chemotherapy regimens, it is important to investi- Not applicable gate alternative approaches to improve ALL outcome without adding additional chemotherapy agents. Competing interests The authors declare that they have no competing interests. Conclusions Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in Switching obese mice with ALL from a high-fat to published maps and institutional affiliations. low-fat diet substantially improved survival with VCR treatment, though not with L-asparaginase or dexa- Author details Diabetes and Obesity Program, Center for Endocrinology, Diabetes and methasone treatment. The finding that a dietary inter- Metabolism, Children’s Hospital Los Angeles, Keck School of Medicine, vention can improve ALL treatment outcome in a 2 University of Southern California, Los Angeles, CA, USA. Children’s Center for preclinical model should be further studied so that its Cancer and Blood Diseases, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. Present potential benefit can be evaluated in both obese and Address: Cure 4 The Kids Foundation, Las Vegas, NV, USA. Division of non-obese patients. Pediatric Endocrinology, UCLA Children’s Discovery and Innovation Institute, David Geffen School of Medicine UCLA, 10833 Le Conte Ave, Los Angeles, CA 90095-1752, USA. Present Address: Aptose Biosciences, San Diego, CA, USA. Additional files Received: 26 July 2018 Accepted: 28 September 2018 Additional file 1: Figure S1. Serum reduction does not improve efficacy of Ara-C or DNR in vitro. A–C Viable 8093 (A), BV173 (B), and Nalm6 (C) cells after 72 h exposure to chemotherapy Ara-C (left) or DNR (right) N =4. References (TIF 979 kb) 1. Calle EE, Rodriguez C, Walker-Thurmond K, Thun MJ. Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. N Additional file 2: Figure S2. Dietary restriction does not affect ALL Engl J Med. 2003;348(17):1625–38. outcome in xenograft NSG model treated with vincristine, dexamethasone, 2. Butturini AM, Dorey FJ, Lange BJ, Henry DW, Gaynon PS, Fu C, et al. Obesity and L-asparaginase (VDL). 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J Clin Oncol. 2016;34(22):2591–601. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cancer & Metabolism Springer Journals

Switch to low-fat diet improves outcome of acute lymphoblastic leukemia in obese mice

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Springer Journals
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Copyright © 2018 by The Author(s).
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Biomedicine; Cancer Research; Oncology; Metabolomics; Metabolic Diseases; Imaging / Radiology; Cell Biology
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2049-3002
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10.1186/s40170-018-0189-0
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Abstract

Background: It is becoming increasingly recognized that weight and nutritional status can impact cancer survival. We have previously shown that obese mice with syngeneic acute lymphoblastic leukemia (ALL) have poorer response to chemotherapy treatment than control mice. We therefore investigated whether dietary intervention could improve outcome from the most common pediatric cancer, ALL. Methods: Diet-induced obese (DIO) mice raised on a 60% calories from fat diet and control mice were implanted with syngeneic ALL cells. Some DIO mice were switched to the low-fat control diet. Survival from ALL was assessed without or with chemotherapy treatment starting at the time of the diet switch. Cells from DIO mice before and after diet switch were assessed by FACS for BrdU incorporation and phosphorylation status of AKT, S6K, and EIF2a. Similar experiments were done with human ALL xenografts. Mouse and human ALL cells were cultured in media with 10% or 5% fetal bovine serum, and sensitivity to chemotherapies assessed. Results: DIO mice had poorer survival (17%) after vincristine monotherapy than control mice on a 10% low fat diet (42%; n = 12/group; p = 0.09, log rank). However, switching obese mice to the low-fat diet prior to initiation of vincristine led to dramatically improved survival (92%, p < 0.01 vs both other groups). In vitro, FBS restriction made murine and human ALL cells more sensitive to vincristine. Interestingly, while serum restriction enhanced ALL sensitivity to dexamethasone and L-asparaginase, dietary switch did not improve survival of DIO mice treated with either drug in monotherapy. Thus, it appears that dietary intervention has a unique effect to improve ALL cell sensitivity to vincristine in vivo. Conclusions: We report herein that a dietary intervention can improve ALL outcome in a preclinical model. Further work is needed to identify the mechanisms of this effect and investigate potential impact on human leukemia in patients. Keywords: Obesity, Adipose tissue, Dietary intervention, Chemotherapy, Caloric restriction Background (HR-ALL) by 50% [2], a finding confirmed in meta-analysis Obesity increases the incidence of many cancer types, and [3]. In a separate cohort, we reported that obese children obese cancer patients have a higher risk of mortality from were 2.74 times more likely to be minimal residual disease their disease [1]. In 2007, a retrospective review of two large (MRD) positive [4], which portends increased relapse risk cohorts demonstrated that obesity at the time of diagnosis and reduced event-free survival [5]. Moreover, recent stud- increases risk of relapse in children with National Cancer ies show 1 in 3 children with ALL were overweight or obese Institute/Rome High-Risk acute lymphoblastic leukemia at diagnosis [6, 7]. Much attention has been given to the role of diet on cancer outcomes. Meta-analysis has shown that cancer * Correspondence: smittelman@mednet.ucla.edu survivors who maintain high quality diets have lower over- Diabetes and Obesity Program, Center for Endocrinology, Diabetes and all mortality, though no significant reduction in cancer Metabolism, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA recurrence [8]. Less clear is whether dietary intervention Division of Pediatric Endocrinology, UCLA Children’s Discovery and during cancer treatment might improve outcome. Caloric Innovation Institute, David Geffen School of Medicine UCLA, 10833 Le Conte restriction has garnered much attention in this realm, as it Ave, Los Angeles, CA 90095-1752, USA Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tucci et al. Cancer & Metabolism (2018) 6:15 Page 2 of 8 can potently reduce insulin-like growth factor 1 (IGF-1), a Mouse models stimulator of cellular metabolism through activation of the High fat diet-induced obese (DIO) and control C57Bl/6J PI3K/Akt/mTOR axis [9, 10]. Non-transformed cells mice were purchased from Jackson Laboratories (Bar respond to this low nutrient state by downregulating Harbor, MI, USA). Mice had been raised on either a 60% mTOR activity and entering a state of quiescence, which (obese) or 10% (control) calories from fat diet (Research can protect them from some chemotherapies; however, Diets, D12492 and D12450B, respectively). Because cancer cells often exhibit constitutive activation of PI3K/ female mice do not become as significantly obese in this Akt/mTOR [11]. This constitutive activation cannot be model and are not available as DIO mice from Jackson modulated by nutrient restriction, leading to retained or in- Laboratories, only male mice were used for these stud- scid tm1Wjl creased chemosensitivity of cancer cells [12, 13]. Additional ies. Male NSG mice (NOD.Cg-Prkdc Il2rg /SzJ) mechanisms also likely contribute to the beneficial effects are NOD/SCID IL2-receptor gamma chain knockout of caloric restriction, including reduced fuel availability, re- mice commonly used for xenograft. NSG mice were duction of inflammation, and lower oxidative stress [9, 14]. made obese using selective culling and a high-fat diet as We have developed a mouse model which recapitu- previously described [19]. lates the clinical observation that obese children have worse ALL outcome; high-fat diet-induced obese (DIO) ALL survival experiments mice implanted with syngeneic ALL had a poorer sur- ALL implantation experiments were performed on DIO vival outcome after treatment with either vincristine and control mice at ~ 20 weeks of age. At this time, (VCR) or L-asparaginase [15, 16]. However, whether the 10,000 GFP pre-B-cell ALL 8093 cells were implanted effects of obesity on ALL outcome in mice or patients is retro-orbitally. 6 or 7 days after ALL implantation, reversible remains unknown. While caloric restriction depending on the experiment, DIO mice were random- has been shown to improve chemotherapy efficacy in ized to continue on their high-fat diet or switched to the mouse models of solid tumors [12], it has not been control diet (10% calories from fat, provided ad libitum). tested in the most common childhood cancer, acute In some experiments, monotherapy with vincristine lymphoblastic leukemia. Therefore, we designed the (0.5 mg/kg/week intraperitoneal) was started on day 7, present study to test whether a dietary intervention and this dose was adjusted for body weight each week. could improve ALL outcome in obese mice. Similar experiments were performed with L-asparaginase (800 IU/kg/day, Monday–Friday) or dexamethasone (8 mg/kg/day, Monday–Friday). In other experiments, Methods DIO and control NSG mice were implanted with LAX7 Cell culture cells, and after a 17 day engraftment period, half of the Murine pre-B ALL cells were previously isolated from a DIO mice switched to control diet. On day 18, treatment BCR/ABL transgenic mouse (“8093 cells” [17]) and with vincristine, L-asparaginase and dexamethasone transduced with GFP. Human leukemia cell lines in- (same doses as above) was started and continued for cluded BV173 (pre B Ph + ALL, ATCC) and Nalm-6 (B 4 weeks. Mice in all survival experiments were moni- cell precursor leukemia, ATCC). Cells were authen- tored daily for food intake and body weight, and onset ticated by the University of Arizona Genetics core in of progressive leukemia (paralysis, hunched posture, November 2016 and tested negative for mycoplasma. palpable mass > 1 cm, poor grooming, etc.), at which Eight thousand ninety-three cells were cultured in time they were euthanized. McCoy’s 5A media (Invitrogen), supplemented with 1 mM sodium pyruvate, 2 mM Glutamax, 10 μg/mL Tissue harvesting and flow cytometry gentamycin and either 5% or 10% FBS (Denville Scientific In some experiments, mice were anesthetized with keta- or Omega Scientific). Human cell lines were cultured in mine/xylazine and perfused with PBS for organ harvest RPMI 1640 (Invitrogen), supplemented as above. at various timepoints before and after ALL implantation. Chemotherapy sensitivity experiments were performed When performing cell cycle analysis, mice were injected over 72 h, with surviving viable cells counted by with BrdU intraperitoneally 4 h prior to organ harvest. blinded observers in triplicate using trypan blue exclu- Spleen and femurs were removed, and spleen pulp and sion manually, or with a Countess II (ThermoFisher). femoral marrow were extruded with red blood cell lysis LAX7 cells are patient-derived ALL cells with normal buffer (BD Biosciences, San Jose, CA, USA) for flow cy- karyotype which were expanded by passage through tometry. Red blood cell-free bone marrow and spleen NSG mice (mice described below) and used for xeno- pulp were filtered through 40 μm cell strainers to create graft [18]. Cells were cultured on OP-9 stroma cells single cell suspensions. Cells utilized for cell cycle ana- with αMEM supplemented with 20% FBS, 100 IU/mL lysis were processed according to the manufacturer’s penicillin, and 100 μg/mL streptomycin. protocol (APC BrdU Flow Kit, BD Biosciences). Cells Tucci et al. Cancer & Metabolism (2018) 6:15 Page 3 of 8 not used for cell cycle analysis were fixed in 4% PFA at relative sparing of carbohydrate intake, due to the in- 37 °C for 10 min and then permeabilized in chilled creased carbohydrate density of the control diet. After a methanol for 30 min. Cells were washed and incubated couple of weeks, food intake in the switched mice in- in Mouse Fc Block (BD Biosciences) for 15 min then in- creased, and nutrient intake became similar to that of cubated for 1 h in the following fluorophore-conjugated controls. antibodies: pEIF2α(Ser51)-PE/Cy7, pAKT(Ser473)-A647, Dietary restriction in animals leads to a large number pS6K(Thr412)-PE (Bioss USA, Boston, MA, USA). Cells of hormonal, physical, physiologic, and metabolic were analyzed using the LSR II Flow Cytometer (BD changes that cannot be completely modeled in vitro. Biosciences) in the CHLA Flow Cytometry Core. Cells However, FBS restriction can simulate some of the de- were initially gated to exclude DAPI-positive dead cells cline in growth factors that are observed with caloric re- and forward and side scatter to exclude debris. Gating striction and weight loss. Reducing FBS concentration on GFP allowed analysis of ALL vs. host cells. increased VCR cytotoxicity (Fig. 2a); however, FBS con- centrations below 5% impaired cell growth and viability. We therefore used 5% FBS, which did not affect their Data analysis proliferation rate but made them significantly more sen- Kaplan Meier survival curves were generated and com- sitive to VCR (Fig. 2b, c). Similar VCR sensitization was pared using Cox regression. Food intake was measured observed in human ALL cell lines as well, BV173 and manually by mouse cage, and reported as daily averages Nalm6 (Fig. 2d–f). or three-day moving averages as described in results. Vi- able cells and proportions of cells labeled with BrdU or Diet restriction does not alter ALL response to L- other antibodies were compared between diet switch asparaginase or dexamethasone in vivo and DIO mice using two-sided, paired t-tests. EC was Similar ALL survival experiments were done as above, calculated by fitting normalized dose-response data to but using L-asparaginase or dexamethasone monother- the equation: %viable cells ¼ . All ana- ðLogðdoseÞ−LogðEC50ÞÞ 1þ10 apy starting on day 7. Diet switch had no detectible lysis was performed using GraphPad Prism and Micro- effect on survival in these experiments (Fig. 3a, b). FBS soft Excel. restriction did increase 8093 cell sensitivity to dexa- methasone (EC 9.1 ± 2.3 vs. 16.3 ± 3.7 nM, p = 0.02) Results and tended to increase sensitivity to L-asparaginase Diet restriction sensitizes ALL cells to vincristine (EC 0.80 ± 0.22 vs. 1.30 ± 0.21 IU/mL, p =0.13; Fig. 3c, d), To determine whether switching diet could improve ALL but not to Ara-C or daunorubicin (Additional file 1:Figure treatment outcome, we implanted 16 DIO and 8 S1). In addition, survival experiments performed with hu- non-obese control mice with syngeneic ALL cells (day 0; man ALL xenografted NSG mice treated with a combin- Fig. 1a). On day 7, half of the DIO mice were switched to ation of vincristine, L-asparaginase, and dexamethasone the low-fat control diet, while the rest were continued on (VDL)showedthatdiet-restricted mice hadnodifference their regular diet (final n = 8 per group). Time to progres- in survival from DIO or control mice (Additional file 2:Fig- sive ALL was not affected by diet in these mice (Fig. 1b). ureS2).Thus, theeffect of dietaryrestriction to sensitize In other groups of mice, VCR was started on day 7, and ALL to chemotherapy treatment in vivo appears to be rela- VCR dose adjusted weekly based on body weight. In this tively specific to VCR. experiment, DIO mice switched to the low-fat diet had the best survival, which was significantly better than mice Diet restriction does not alter cell cycle rate of host or maintained on the high-fat diet (p < 0.001), and even bet- ALL cells ter than control mice that had been raised on the low-fat To investigate whether dietary restriction changes cell diet (p <0.01; n = 6 repeated in two separate experiments sensitivity to VCR by altering ALL or host cell cycling for final n = 12 per group; Fig. 1c). Overall survival at the rate, cells were harvested from groups of DIO mice be- end of the experiment was 92% in the diet-switch group, fore or after diet switch, 4 hours after BrdU injections. 42% in the control group and 17% in the DIO group. ALL and host cells were separated by FACS from Diet-switched mice in additional experiments exhibited spleens and bone marrows of DIO mice on day 6 after rapid weight loss, where their body weight declined to ALL implantation (before any diet switch), day 8 (1 day match those of the control mice within approximately after diet switch), or day 14 (7 days after diet switch). 6days (Fig. 1d). This was associated with a substantial de- The percentage of GFP+ ALL cells in S phase were de- cline in food intake, despite access to ad libitum feed. termined and compared to GFP-host cells (splenocytes Switched mice reduced food intake for several days after or marrow cells) from the same tissues. The percentage the diet switch, leading to substantially decreased intake of cells in S phase was higher in splenic ALL cells than of total calories, protein and fat (Fig. 1e). There was a non-ALL cells, but similar between ALL and non-ALL Tucci et al. Cancer & Metabolism (2018) 6:15 Page 4 of 8 Fig. 1 Dietary restriction improves obese leukemic C57BL/6J mouse survival after vincristine treatment. a Scheme showing diet intervention in survival experiments. b Survival of mice with ALL in each diet group with no chemotherapy treatment (n = 12/group). c Survival of mice with ALL in each diet group treated with VCR. ▼ indicate VCR doses. Vehicle-treated mice were distributed evenly between all three diet groups, and were combined due to no difference in survival. N = 12/group; **p < 0.01, ***p < 0.001, log rank. d Body weight of mice on each diet. e Total (left), carbohydrate (center), and fat (right) calorie daily intake in mice from each diet group. Intake is shown as daily average up until day 5 after the diet switch, and as 3 point moving average thereafter, due to high day-to-day variability. Standard deviations reflect variance between 2 and 4 cages, not individual mice. Since protein content was identical between diets, the relative intake was proportional to total caloric intake and not shown separately cells from marrow (Additional file 3: Figure S3). Diet samples (Additional file 3: Figure S3), confirming the switch did not significantly alter this percentage in either higher metabolic rate in cancer cells. However, diet switch cell type. had no significant effect on phosphorylation state of any of these proteins in host or ALL cells. Diet restriction does not alter AKT signaling in host or ALL cells Discussion To determine how diet restriction might alter relevant We report herein that switching obese mice from a intracellular signaling pathways, spleen- and marrow-de- high-fat to a low-fat diet improves ALL survival with rived cells from obese and obese-dieted mice (described vincristine treatment from 17 to 92%. This is the first above) were labeled with antibodies for EIF2a/pEIF2a, study of which we are aware testing a diet intervention AKT/pAKT, and S6K/pS6K and analyzed by flow cytome- on treatment outcome from a hematological malignancy. try. ALL cells from both tissues showed higher phosphor- Other studies have evaluated whether dietary interven- ylation levels of these proteins than host cells in most tion can affect treatment outcome of solid cancers. Tucci et al. Cancer & Metabolism (2018) 6:15 Page 5 of 8 Fig. 2 Serum restriction sensitizes ALL to vincristine treatment. a Viable 8093 cells after 72 h in culture media with various concentrations of FBS, alone, or with 10 nM VCR (n = 3). b Dose response of murine ALL 8093 cells cultured with VCR in 5 or 10% FBS (n = 4). c Viable 8093 cells after culture alone or with 10 nM VCR (n = 7) ***p < 0.001, two-sided, paired t test. d EC values calculated from dose responses of vincristine with all three cell lines; connecting lines show paired experiments. *p < 0.05, ***p < 0.001, two-sided, paired t test. e, f Dose response of human ALL cell lines BV173 (e) and Nalm6 (f) with VCR in 5 or 10% FBS (n =4) Fig. 3 Diet restriction does not sensitize murine ALL cells to dexamethasone or L-asparaginase. a, b Survival of mice in each diet group treated with dexamethasone (a, n =5–6) or L-asparaginase (b, n = 6). c, d Viable cells after 72 h in culture media with 5 or 10% FBS, alone or with 70 nM dexamethasone (c) or 2.5 IU/mL L-asparaginase (d), n =3 Tucci et al. Cancer & Metabolism (2018) 6:15 Page 6 of 8 Switching DIO mice from a high-fat to low-fat diet, which would require extensive additional experiments similar to the present study, has been shown to improve and should be evaluated in future studies. survival from melanoma in mice treated with dacarba- It is also possible that the protective effect of a dietary zine [20]. Ketogenic diets, containing very low carbohy- switch is mediated in part by immunological mecha- drates, are used for patients with intractable epilepsy nisms. In the 2010 update to the well-known “Hallmarks and cause decreases in blood glucose concentrations but of Cancer,” four new principles were identified as vital to elevations in circulating free fatty acids and ketones [21]. carcinogenesis and cancer cell survival, one being eva- These diets have been shown to have an anti-tumor ef- sion of immune destruction [25]. In addition to their fect in 12 separate murine cancer models [22] and are direct cytotoxic effect, some chemotherapies have been being evaluated in patients. Fasting and short-term star- identified as inducers of immunogenic cell death, vation has also been shown to improve treatment out- whereby their mechanism of cytotoxicity invokes a host come from a wide variety of solid tumors in mice [12]. T-cell-mediated immunologic response [26]. These in- How efficacious these diets will be in cancer patients re- clude the anti-microtubule agents docetaxel and pacli- mains to be seen. taxel, as well as the anthracyclines, daunorubicin and Our findings are consistent with retrospective data doxorubicin, and the anthracendione, mitoxantrone. which shows that reversing obesity may be associated Interestingly, caloric restriction and short-term fasting with improved ALL outcome in youth. Orgel et al. ob- have been shown to improve T-cell anti-tumor reactivity served that ALL patients who had spontaneously lost and reduce immune-suppressing T-regs. Combined, weight and changed from obese to non-obese category these studies suggest that dietary restriction could syner- over their treatment had better outcome than those who gize with a certain subset of chemotherapies to augment remained obese for > 50% of their treatment course [23]. host anti-tumor response and improve treatment out- Based on this observation, and our current findings, we comes. This would be consistent with our finding that have launched a clinical intervention trial of mild dietary NSG mice, which lack immune systems, obtained no restriction and increased physical activity in children benefit from the dietary switch. On another note, it is with newly diagnosed ALL (IDEAL Weight in ALL Trial; particularly interesting that three of the other four iden- NCT 02708108). This and similar studies are critical to tified immune inducers, daunorubicin, doxorubicin, and help determine which cancers respond to dietary inter- mitoxantrone, are commonly used in primary and re- ventions and which specific interventions work for lapse ALL chemotherapy regimens. These chemothera- which cancers. peutic agents were not studied in our current model but While we were unable to identify the mechanism should be tested in future studies. whereby dietary restriction improved ALL outcome in Our study has several limitations. Perhaps our biggest our study, we did rule out some important mecha- limitation is that we did not detect an effect of dietary nisms. VCR cytotoxicity is cell cycle dependent, as op- intervention in our human xenograft model. This limits posed to L-asparaginase and dexamethasone, which the degree to which our results may be directly translat- could explain why mice treated with dexamethasone able to human leukemia. The lack of effect in this model or L-asparaginase derived no survival benefit from a could have been due to the triple chemotherapy regimen dietary switch. However, we did not detect an effect of used, which could have masked a small effect of vincris- dietary intervention on cell cycle of ALL or host cells tine alone, to the immunodeficient state as discussed one or 7 days after diet initiation. We also found no above, or to other mouse strain differences. In any case, effect of diet intervention on the ALL or host cell ac- dietary intervention in human xenograft models should tivity of pathways commonly evaluated for effects of be further evaluated so that relevance to human dietary intervention on cancer, though we did observe leukemia can be confirmed. Another limitation is that an increased activity of these pathways in the cancer we tested a relatively crude dietary intervention—switch- cells vs. host cells. Lack of effect of diet on AKT or ing DIO mice from a high-calorie/high-fat diet to a S6K phosphorylation implies that our intervention lower-calorie/low-fat diet. This switch induced several was not acting through IGF-1 or mTOR, respectively. changes, including decreased calorie, protein, and fat in- eIF2a phosphorylation is observed with amino acid take, while sparing carbohydrate intake. These changes deprivation, oxidative stress, and unfolded protein re- undoubtedly altered the animals’ insulin sensitivity, cir- sponse [24], arguing against these potential mecha- culating growth factors, body composition, and fuel nisms of our dietary intervention. It is possible that availability. Indeed, another study which utilized a simi- these signals may have mediated effects after 7 days, lar high-fat to low-fat diet switch in C57Bl/6 mice which we would have missed with our experimental showed a number of physiological effects, including design. Further, while these findings argue against decreases in glucose and insulin, improvement in lipids these pathways, we did not exhaustively rule them out, and free fatty acids, and decrease in several pro- Tucci et al. Cancer & Metabolism (2018) 6:15 Page 7 of 8 inflammatory cytokines and adipokines [20]. This broad Acknowledgements The authors would like to thank Ehsan Ehsanipour for assistance in range of effects may contribute to the efficacy of this performing these studies. intervention, but at the same time makes identification of specific mechanisms difficult. Thus, further study will Funding This work was supported by R01CA201444 (SDM), R01CA213129 (SDM), need to be done to determine which of these changes R01CA172896 (YMK), and funds from The Saban Research Institute, CHLA. and effects contribute to the improved survival. On the other hand, murine obesity induced by a high-fat diet is Availability of data and materials The datasets used and/or analyzed during the current study are available not completely analogous to human obesity, which is from the corresponding author on reasonable request. generally associated with excess carbohydrate intake; thus, utility of these models for this purpose may be lim- Authors’ contributions JT, WA, and TC designed and performed experiments, analyzed and ited. Finally, despite the large observed effect of dietary interpreted data, and helped in the preparation of the manuscript. XS helped intervention on vincristine efficacy, the mechanism be- perform some experiments and develop flow cytometry experiments. YMK hind this effect remains elusive. provided patient sample cells and advised on xenograft model experiments. SDM conceived of the experiments, oversaw all aspects of the study, and While treatment of childhood ALL has led to substantial wrote the manuscript. All authors approved the final manuscript. improvements in survival, there are still ~ 8% children who relapse from this disease every year [27]. In addition, Ethics approval All studies were approved by the CHLA IACUC and performed in accordance children with certain ALL subtypes, adolescents and with the US Public Health Service Policy on Humane Care and Use of adults, and obese patients are all at increased risk of Laboratory Animals. relapse and mortality. Given the substantial toxicity of Consent for publication current chemotherapy regimens, it is important to investi- Not applicable gate alternative approaches to improve ALL outcome without adding additional chemotherapy agents. Competing interests The authors declare that they have no competing interests. Conclusions Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in Switching obese mice with ALL from a high-fat to published maps and institutional affiliations. low-fat diet substantially improved survival with VCR treatment, though not with L-asparaginase or dexa- Author details Diabetes and Obesity Program, Center for Endocrinology, Diabetes and methasone treatment. The finding that a dietary inter- Metabolism, Children’s Hospital Los Angeles, Keck School of Medicine, vention can improve ALL treatment outcome in a 2 University of Southern California, Los Angeles, CA, USA. Children’s Center for preclinical model should be further studied so that its Cancer and Blood Diseases, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA. Present potential benefit can be evaluated in both obese and Address: Cure 4 The Kids Foundation, Las Vegas, NV, USA. Division of non-obese patients. Pediatric Endocrinology, UCLA Children’s Discovery and Innovation Institute, David Geffen School of Medicine UCLA, 10833 Le Conte Ave, Los Angeles, CA 90095-1752, USA. Present Address: Aptose Biosciences, San Diego, CA, USA. Additional files Received: 26 July 2018 Accepted: 28 September 2018 Additional file 1: Figure S1. Serum reduction does not improve efficacy of Ara-C or DNR in vitro. A–C Viable 8093 (A), BV173 (B), and Nalm6 (C) cells after 72 h exposure to chemotherapy Ara-C (left) or DNR (right) N =4. References (TIF 979 kb) 1. Calle EE, Rodriguez C, Walker-Thurmond K, Thun MJ. Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults. N Additional file 2: Figure S2. Dietary restriction does not affect ALL Engl J Med. 2003;348(17):1625–38. outcome in xenograft NSG model treated with vincristine, dexamethasone, 2. Butturini AM, Dorey FJ, Lange BJ, Henry DW, Gaynon PS, Fu C, et al. Obesity and L-asparaginase (VDL). 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Cancer & MetabolismSpringer Journals

Published: Nov 1, 2018

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