Presentation on theme: "The eIF2 kinase GCN2 is essential for the murine immune system to adapt to amino acid deprivation by asparaginase. Piyawan Bunpo, Judy K. Cundiff, Rachel."— Presentation transcript:
The eIF2 kinase GCN2 is essential for the murine immune system to adapt to amino acid deprivation by asparaginase. Piyawan Bunpo, Judy K. Cundiff, Rachel B. Reinert, Ronald C. Wek, Carla J. Aldrich, and Tracy G. Anthony
Authors and Acknowledgments This work was supported by: American Institute for Cancer Research Indiana University School of Medicine National Institutes of Health R01GM49164 We are grateful for the technical assistance provided by: Diana Fuqua Debbie Wagner Gary White P. BunpoJ. CundiffR. ReinertR. WekC. AldrichT. Anthony
Bone marrow from child with B-cell ALL Asparaginase and Treatment of Acute Lymphoblastic Leukemia Asparaginase (ASNase) is an integral part of the multi-drug induction regimen for acute lymphoblastic leukemia, the most common childhood cancer. Adverse effects can complicate treatment, compromise success. Includes: Hepatic dysfunction Pancreatitis Immunosuppression Bone marrow from child with T-cell ALL Images taken from: http://emedicine.medscape.com/article/990113-diagnosis
Asparaginase Depletes Asparagine aspartate glutamineglutamate asparagine ATP AMP + PPi ASNS ASNase aspartate + NH 3 glutamate + NH 3 ASNase Lymphoblasts and lymphocytes depend on exogenous asparagine for growth. (Ann Rev Biochem 75:629-654, 2006) Drug-induced amino acid starvation
Diverse Stressors in Mammals are Integrated via Phosphorylation of eIF2 Increased translation of genes with uORFs (e.g., ATF4) Decreased general mRNA translation PEK (PERK) Viral Infection PKR Heme Deprivation Heat Shock HRIGCN2 Amino Acid Deprivation ER Stress eIF2 P
Asparaginase Activates Amino Acid Deprivation Responses Asparaginase GCN2 p-eIF2 ATF4 translation Transcription of genes involved in: Amino acid metabolism (ASNS) Apoptosis (CHOP) p-4EBP1 p-S6K1 General mRNA translation Liver onlyLiver and Spleen Summarized from: J. Biol. Chem. 281:31222, 2006 J. Biol. Chem. 284: 32742, 2009
Asparaginase Reduces Lymphocyte Populations in Mice Data summarized from: J. Nutr. 138:338, 2008 Thymus: CD4- CD8- CD4+ CD8+ CD4- CD8+ CD4+ CD8- Bone Marrow: B220+ sIgM- B220+ sIgM+ Spleen: CD4+ CD8+ CD19+ CD3+ Tissue weight Total cell number Tissue weight Total cell number
Determine the role of GCN2 in the mechanism by which asparaginase causes immunosuppression. Study Aim
Hypotheses Amino acid starvation by asparaginase activates GCN2. GCN2 functions to alleviate or adapt to cell stress by amino acid deprivation. Loss of GCN2 function renders the immune system more sensitive to the cytotoxic effects of amino acid deprivation, enhancing cell death of lymphocytes.
Experimental Design Subjects ◦ Wild-type C57BL6/J mice (GCN2 +/+ ) ◦ C57BL6/J mice with whole body GCN2 deletion (GCN2 -/- ) Treatments ◦ Asparaginase - Effective Dose (3 IU/g) vs. saline for 2-6 d ◦ Asparaginase - Lower Dose (<2 IU/g) vs. Inactivated for 7 d Time Points ◦ Tissues collected on days 2, 3, 4, 6, or 7 Measured Outcomes
Loss of GCN2 exacerbates reductions in wet weight and loss of total cell numbers in thymus and spleen by asparaginase. Figure 1 and Supplemental Figure 1 above GCN2 +/+ PBS GCN2 +/+ ASNase GCN2 -/- ASNase GCN2 -/- PBS 1 cm A. 1 cm PBSASNase PBS GCN2 +/+ GCN2 -/- B.
Asparaginase more effectively depletes total cell numbers in the bone marrow and mesenteric lymph nodes in GCN2 -/- mice. Table 4 and Supplemental Figure 3 above GCN2 +/+ GCN2 -/-
Asparaginase More Aggressively Reduces Maturing Populations of Thymocytes in GCN2 -/- Mice versus Wild-Type Mice. Table 2 and Supplemental Table 3 above GCN2 +/+ GCN2 -/- cells x 10 6 HIALD ASNaseHIALD ASNase Total79.00 ± 6.9775.78 ± 12.31 *107.30 ± 12.7953.85 ± 7.96 * CD4- CD8-2.15 ± 0.332.58 ± 1.032.63 ± 0.192.48 ± 0.31 CD4+ CD8+65.41 ± 5.78 ab 61.22 ± 18.61 * b 88.02 ± 11.31 a 41.03 ± 6.65 * c CD4+ CD8-9.02 ± 1.049.29 ± 2.34 *12.67 ± 1.467.55 ± 1.11 * CD4- CD8+2.97 ± 0.382.68 ± 0.80 *3.56 ± 0.461.47 ± 0.15 * Double positive thymocytes most negatively impacted.
Thymic cell death is amplified in GCN2 -/- mice treated with asparaginase, resulting in extensive fatty replacement in thymi. GCN2 +/+ PBS GCN2 +/+ ASNase GCN2 -/- ASNase GCN2 -/- PBS 10 m sections at 200X magnification GCN2 +/+ PBS GCN2 +/+ ASNase GCN2 -/- ASNase GCN2 -/- PBS 4 days A. B. Figures 2B and 3 and Supplemental Figure 2 above TUNEL Assay Oil Red O
Asparaginase Reduces Both Major T cell Populations (CD4+, CD8+) in the Spleen to a Much Greater Extent in GCN2 -/- Mice. Table 3 Asparaginase Reduces CD11b+ cells in Spleen to a Greater Extent in GCN2 -/- Mice. Table 4
Activation of eIF2(P)-mediated events by asparaginase are precluded in the spleens and thymi of GCN2 -/- Mice. Supplemental Figure 4 A. B. GCN2 +/+ GCN2 -/- PPAA GCN2 +/+ GCN2 -/- PPAA GCN2 +/+ GCN2 -/- PPAA GCN2 +/+ GCN2 -/- PPAA D. C. p-4EBP1 p-S6K1 Thymus Spleen Phosphorylation events downstream of mTORC1 are reduced in the Spleens and Thymi of GCN2 -/- Mice. Figures 2 and 4
Summary and Conclusion Loss of GCN2 enhances immunosuppression by asparaginase. Emphasizes the essential role for GCN2 in the ability of the immune system to adapt to amino acid stress.
Significance GCN2 plays a larger role in the immune system beyond regulating T helper cell function. Targeted inhibition of GCN2 may render a hyperproliferating lymphocyte much more sensitive to lower doses of asparaginase. This could reduce overall cytotoxicity and complications associated with asparaginase. The integrated stress response is important to study with respect to asparaginase and similar drugs used to treat cancer.
For additional questions and comments, please contact: Tracy G. Anthony, Ph.D. firstname.lastname@example.org 812-465-1199 Thank you for watching this pubcast!