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, Ph.D.tganthon@iupui.edu

812-465-1199

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