Experimental Design and Methods Abstract ...

Loss of Activating Transcription Factor 4 (ATF4) Alters the Homeostatic Amino Acid Response (AAR) in the Liver of Mice Treated with Asparaginase Rana J.T. Al-Baghdadi1, Emily T. Mirek2, Lindsey A. Phillipson-Weiner2, Yongping Wang2, William J. Belden1, Ronald C. Wek3 and Tracy G. Anthony 1,2,3. 1Endocrinology and Animal Biosciences and 2Nutritional Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901; 3Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202

Experimental Design and Methods Study Subjects: 8 wk old male and female C57BL/6J mice Freely–fed commercial diet

Treatment Groups: WP: ATF4+/+ mice injected with phosphate buffered saline WA: ATF4+/+ mice injected with L-asparaginase HP: ATF4+/- mice injected with phosphate buffered saline HA: ATF4+/- mice injected with L-asparaginase KP: ATF4-/- mice injected with phosphate buffered saline KA: ATF4-/- mice injected with L-asparaginase

Strains: Wild type (ATF4+/+ ) Heterozygous (ATF4+/- ) Knockout (ATF4-/- )

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2 3 4 5 6 7 Once a day i.p. injection of Elspar® L-asparaginase Dose: 3 I.U. per gram body weight or equivolume PBS

3 days acclimation Singly housed

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B o d y W e ig h t C h a n g e

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Neutral lipids are represented by red dots. Images were taken at 20X magnification and represent 3-5 mice per treatment group.

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ATF4 independent AAR gene induction

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ATF4 deficiency would compromise the hepatic AAR to asparaginase and augment hepatotoxicity.

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TUNEL positive cells are indicated as dark blue dots. Images were taken at 20X magnification and represent 35 mice per treatment group.

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ATF4 mediates many of the GCN2-driven hepatic adaptive responses to asparaginase which prevents liver inflammation and cell damage.

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References

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1. Rytting, M. Role of L-asparaginase in acute lymphoblastic leukemia: focus on adult patients. Blood and Lymphatic Cancer: Targets and Therapy 2:117-124, 2012.

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2. Dores GM, Devesa SS, Curtis RE, et al. Acute leukemia incidence and patient survival among children and adults in the United States, 2001-2007. Blood 119: 34-43, 2012.

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4. Wek RC, Jiang HY, Anthony TG. Coping with stress: eIF2 kinases and translational control. Biochem Soc Trans 34: 7-11, 2006.

C H O P m R N A e x p r e s s io n

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Hypothesis

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Conclusions

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ATF4

Amino Acid synthesis

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3. Masuoka HC, Townes TM. Targeted disruption of the activating transcription factor 4 gene results in severe fetal anemia in mice. Am Soc Hematology 99: 736-745, 2002.

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Cell fate

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ATF4 dependent AAR gene induction

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Oxidative defenses

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Figure 6. Loss of ATF4 compromises oxidative defenses and promotes inflammation following asparaginase treatment. A. a

Figure 4. Livers from ATF4-/mice show widespread cell damage and death following asparaginase treatment.

Figure 2. Loss of ATF4 precluded induction of some not all hepatic AAR genes induced by asparaginase. Ser51

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Figure 1. treated with asparaginase demonstrated decreased body weight and earlier onset of morbidity. A. B. C.

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Results

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Protein Synthesis

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Analyses (for detailed description see refs 9, 11): • SDS-PAGE and immunoblotting to evaluate protein expression and phosphorylation status • Quantitative RT-PCR to assess gene expression • Oil Red O stain of frozen liver sections (5 mM) to visualize neutral lipid accumulation • TUNEL assay to evaluate DNA damage and cell death in frozen liver sections (10 mM)

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WP=Wild type + PBS WA=Wild type + Asparaginase Day Day -3 GP=GCN2 -/- + PBS GA=GCN2 -/- + Asparaginase

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Asparaginase (ASNase) is used to treat acute lymphoblastic leukemia (ALL), the most common childhood cancer. It works by degrading asparagine and glutamine, essential for the cancer cell to survive (1). Asparaginase is implicated in many deleterious outcomes such as hepatotoxicity, coagulopathy, pancreatitis and other metabolic complications (2,3). Decreased amino acid levels in the cell is sensed by general control nondepressible 2 (GCN2) (4), a protein kinase that functions to phosphorylate the protein factor eukaryotic initiation factor 2 (eIF2). Phosphorylation of eIF2 during amino acid deficiency activates the amino acid stress response (AAR) pathway (5). Previously our lab showed that GCN2 phosphorylates eukaryotic initiation factor 2 (eIF2) during asparaginase treatment as a defense mechanism to prevent hepatotoxicity and immunosuppression (6, 7, 9, 11). Activation of the AAR inhibits general protein synthesis while at the same time increases mRNA translation of activating transcription factor 4 (ATF4) (8). ATF4 activates transcription of genes involved in oxidative stress, cell cycle control and amino acid metabolism (8). We previously found that GCN2 is necessary for hepatic adaptation to asparaginase (6,9, 11). Maintenance of metabolic homeostasis in the face of nutrient deprivation is largely attributed to the actions of ATF4 in tumors (10). In this study, we endeavored to understand to what extent the asparaginase-induced adaptive AAR is controlled by ATF4. We hypothesized that loss of ATF4 would preclude transcriptional changes induced by asparaginase.

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Figure 5. ATF4 is required for hepatic induction of FGF21 by asparaginase.

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ATF4-/- mice

Background

Figure 3. Asparaginase promoted lipid accumulation in the liver of all mouse strains.

T N F  m R N A e x p r e s s io n

L-asparaginase (ASNase) is widely used to treat leukemia in children but it causes metabolic complications and liver toxicity. ASNase activates the amino acid response (AAR), increasing gene-specific translation of the transcription factor ATF4 to regain homeostasis via altered gene expression. We hypothesized that ATF4 deficiency would compromise the hepatic AAR to ASNase and augment hepatotoxicity. Intact (Atf4wt), heterozygous (Atf4het) and homozygous null (Atf4null) mice were administered ASNase (3 IU/g BW i.p.) or saline once daily for 8d. Blood and tissues were collected 8h after final injection. Atf4null mice treated with ASNase lost body weight and experienced premature mortality while all other strains appeared healthy. ASNase did not alter liver mass but promoted lipid accumulation in all genetic strains of mice to a similar degree. Phosphorylation of eIF2 was increased in the liver of all mice treated with ASNase and in saline-injected Atf4null mice. Examination of the hepatic AAR showed many genes including Asns, Atf5, Chop, Fgf21, 4ebp1 and Gadd34 required ATF4 for full induction. Furthermore, inflammatory stress was evident in the liver of Atf4null but not Atf4het or Atf4wt mice treated with ASNase, similar to that previously reported in Gcn2null mice (AJP 305:E1124, 2013). These results suggest that ATF4 mediates GCN2-driven hepatic adaptive responses to ASNase and prevents liver inflammation but not lipid accumulation. HD070487 (TGA); GM49164 (RCW).

Results F G F 2 1 m R N A e x p re s s io n

Abstract

5. Reinert RB, Oberle LM, Wek SA, Bunpo P, Wang XP, Mileva I, Goodwin LO, Aldrich CJ, Durden DL, McNurlan MA, Wek RC, Anthony TG. Role of glutamine depletion in directing tissue-specific nutrient stress responses to L-asparaginase. J Biol Chem 281:31222-33, 2006. 65 60 55 50 45 40 35 30 25 20 15 10 5 0

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6. Bunpo, P, A Dudley, JK Cundiff, DR Cavener, RC Wek, TG Anthony. GCN2 protein kinase is required to activate amino acid deprivation responses in mice treated with the anti-cancer agent L-asparaginase. J Biol Chem 284:32742-9, 2009. 7. Bunpo P1, Cundiff JK, Reinert RB, Wek RC, Aldrich CJ, Anthony TG. The eIF2 kinase GCN2 is essential for the murine immune system to adapt to amino acid deprivation by asparaginase. J Nutr 140:2020-7, 2010. 8. Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, et al. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell 11:619-33, 2003. 9. Wilson GJ, Bunpo P, Cundiff JK, Wek RC, Anthony TG. The eukaryotic initiation factor 2 (eIF2) kinase GCN2 protects against hepatotoxicity during asparaginase treatment. Am J Physiol Endocrinol Metab 305: E1124-1133, 2013.

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10. Ye J, Kumanova M, Hart LS, Sloane K, Zhang H, De Panis DN, Bobrovnikova-Marjon E, Diehl JA, Ron D, Koumenis C. The GCN2-ATF4 pathway is critical for tumour cell survival and proliferation in response to nutrient deprivation. EMBO J 29: 2082–2096, 2010. HA

Values are means ± S.E.M n=3-12 per group Labeled means without a common letter differ (P< 0.05, ANOVA followed by LSD post hoc).

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11. Wilson GJ, Lennox BA, She P, Mirek ET, Al Baghdadi RJ, Fusakio ME, Dixon JL, Henderson GC, Wek RC, Anthony TG. GCN2 is required to increase fibroblast growth factor 21 and maintain hepatic triglyceride homeostasis during asparaginase treatment. Am J Physiol Endocrinol Metab 308:E283-93, 2015.

Acknowledgements National Institutes of Health grants HD070487 (T.G. Anthony) and GM49164 (R.C. Wek). The authors gratefully acknowledge Gabriel Wilson and Casey Fannell for their expert technical support.