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 ResearchIndiana University School of Medicine
National Institutes of Health R01GM49164
We are grateful for the technical assistance provided by: Diana Fuqua
Debbie WagnerGary White
P. Bunpo J. Cundiff R. Reinert R. Wek C. AldrichT. Anthony
Bone marrow from child with B-cell ALL
Asparaginase and Treatment of Acute Lymphoblastic LeukemiaAsparaginase (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
glutamine glutamate
asparagine
ATP AMP + PPi
ASNSASNase
aspartate + NH3
glutamate + NH3
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)
ViralInfection
PKR
Heme Deprivation Heat Shock
HRI GCN2
Amino AcidDeprivation
ER Stress
eIF2 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-4EBP1p-S6K1
General mRNA translation
Liver onlyLiver and Spleen
Summarized from: J. Biol. Chem. 281:31222, 2006J. Biol. Chem. 284: 32742, 2009
B cells develop in the bone marrowand migrate to the spleen and lymph nodes
T cells mature in the thymus and migrate to the spleen and lymph nodes.
B and T lymphocytes develop from progenitor or stem cells located in the bone marrow.
CD117+(c-kit+)
B220+ B220+sIgM+
CD4+8+CD4-8-
CD4+8-
CD4-8+4+8+ thymocytes undergo
positive and negative selection
B cells
Splenic lymphocytes
Early pro-B Immature B
CD4+8-
CD4-8+
B220+sIgM+CD19+
T cells
in the thymus
Maturation of B and T Lymphocytes
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 weightTotal cell number
Tissue weightTotal 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 DesignSubjects
◦ 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 cmA.
1 cm
PBS ASNase ASNasePBS
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
Series10
5
10
15
20
25PBSASNase
ML
N T
ota
l ce
ll n
um
be
r
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 106 HIA LD ASNase HIA LD ASNase
Total 79.00 ± 6.97 75.78 ± 12.31 * 107.30 ± 12.79 53.85 ± 7.96 *
CD4- CD8- 2.15 ± 0.33 2.58 ± 1.03 2.63 ± 0.19 2.48 ± 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 ± 1.04 9.29 ± 2.34 * 12.67 ± 1.46 7.55 ± 1.11 *
CD4- CD8+ 2.97 ± 0.38 2.68 ± 0.80 * 3.56 ± 0.46 1.47 ± 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 mm 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 4A. B.
GCN2+/+
GCN2-/-
P PA A
GCN2+/+
GCN2-/-
P PA A
GCN2+/+
GCN2-/-
P PA A
GCN2+/+
GCN2-/-
P PA A
D.C.
p-4EBP1
p-4EBP1 p-S6K1
p-S6K1Thymus
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, [email protected]
812-465-1199
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