Targeting glutamine metabolism enhances tumor specific immunity by modulating suppressive myeloid cells Min-Hee Oh, … , Maureen R. Horton, Jonathan D. Powell J Clin Invest. 2020. https://doi.org/10.1172/JCI131859. In-Press Preview Graphical abstract Research Immunology Oncology Find the latest version: https://jci.me/131859/pdf
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Targeting glutamine metabolism enhances tumor specific …€¦ · Glutamine metabolism as a whole is a crucial element of cancer cell metabolism. Glutamine is Glutamine is important
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hydroxyproline and succinate. Surprisingly, of the 200 metabolites queried, kynurenine was the most
differentially regulated. Kynurenine is the product of tryptophan metabolism by IDO and has potent
immunosuppressive effects. IDO knockout mice robustly reject tumors and inhibitors of IDO are being
developed clinically as immunotherapy (36, 56, 57). Unexpectedly, JHU083 inhibited conversion of
tryptophan to kynurenine. However, its mechanism of action was not by directly inhibiting IDO but rather
by inducing the down modulation of IDO gene expression via reduced STAT1 and STAT3 transcriptional
activity.
In addition to inhibiting growth of the primary tumor, glutamine antagonism proved to be potent in
inhibiting the development of metastasis. This observation has important clinical relevance to many tumors
(especially breast cancer) where metastatic spread of the primary tumor negates successful surgical removal.
In the 4T1 model, a major site of metastasis is the lung. Interestingly, we observed both metabolic and
immunologic differences in the lungs of untreated and treated mice even in the absence of macroscopic
metastasis. MDSCs are thought to play an integral role in promoting metastasis (26, 58, 59). It has been
shown that MDSCs increase angiogenesis, tumor invasion, and formation of a pre-metastatic niche by
enhancing pro-angiogenetic factors (such as VEGF, PDGF, b-FGF, and angiopoietins), MMPs, and
chemokines (such as CXCL1, CXCL2, MCP1 and CXCL5) (26). To this end, we observed an increase in
the CD8+ T cells: MDSCs ratio in the lungs of the treated mice even in the absence of observable tumor.
Likewise, kynurenine levels were decreased in the lungs of JHU083 treated mice compared to untreated
mice even before there was evidence of macroscopic metastasis. Previous studies have shown that
kynurenine can promote metastasis by inducing epithelial-to-mesenchymal transition by activating the aryl
hydrocarbon receptor (39).
Immunotherapy in the form of anti-PD1 and anti-CTLA4 has revolutionized our approach to treat
certain cancers. Yet, in spite of these successes it is clear that not all cancers respond to checkpoint blockade
alone and even amongst responsive cancers, not all patients respond (52, 60-62). Such observations point
to multiple different mechanisms of tumor immune evasion. Our data suggest that by targeting glutamine
metabolism we can enhance the efficacy of immunotherapy. To this end, in the anti-PD1 responsive EO771
model, the addition of JHU083 enhanced the overall response of checkpoint blockade. Furthermore, in the
4T1 model that was resistant to combined anti-PD1 and anti-CTLA4 treatment, we could overcome
resistance in part by blocking glutamine metabolism. Overall, these observations support the view that
tumor metabolism represents a means by which cancer cells can evade anti-tumor immune responses.
Further, we provide the preclinical rationale for strategies targeting glutamine metabolism as a means of
enhancing immunotherapy for cancer.
Methods
Further details are provided in the supplemental material.
Animal
C57BL/6J, CD45.1 BALB/cJ, OTI, RAG1 KO, Batf3 KO and BALB/cJ (both male and female mice, 6-8
weeks of age) were purchased from Jackson Laboratories. Mice were randomly assigned to experimental
groups, and ages and genders were matched. NSG mice were obtained from the Johns Hopkins Animal
resources facility. MyD88/TRIF double KO mice were kindly provided by Dr. Franck Housseau (Johns
Hopkins University, Baltimore) (63, 64). TFEB KO mice were kindly provided by Dr. Andrea Ballabio
(Baylor College of Medicine, Houston) (65).
GEO: The RNA sequencing data have been deposited in the GEO under ID codes GSE119733.
Statistics
Graphs were generated and statistical analysis were performed with Prism 7 (GraphPad). Comparison
between two means was done by 2-tailed t-test or non-parametric 2-tailed Mann-Whitney t-test.
Comparisons between three or more means were done by 1-way ANOVA test with multiple comparisons
post-test. Survival test was done by Log-rank (Mantel-Cox) test. The association between two ranked
variables was done by spearman rank correlation. A P value less than 0.05 was considered significant.
Study approval
The Institutional Animal Care and Use Committee of Johns Hopkins University (Baltimore, MD) approved
all animal protocols.
Author Contributions
M.O., I.S., R.L., I.S., W.X., S.L.C., A.J.T., R.L.B., C.H.P., J.E., M.L.A., J.W. and Y.C. performed and
analyzed experiments. L.Z. performed and analyzed LC-MS experiments. P.M., R.R., B.S.S designed and
synthesized JHU083. M.O. and J.D.P wrote the manuscript. M.R.H. and J.D.P. supervised the project.
Acknowledgements
We thank the members of the Horton and Powell labs for review of this manuscript. And we thank Dr. T.C.
Wu for his kind gift of mice. This work was supported by NIH grants (R01CA229451 to J.D.P. and B.S.S.
and S10 OD016374 to the JHU Microscopy Facility), R01HL141490 to M.R.H. and grants R01AI077610,
R01CA226765 to J.D.P.) the Bloomberg∼Kimmel Institute for Cancer Immunotherapy to J.D.P. and B.S.S.,
Under Armour Women’s Health & Breast Cancer Innovation Grants to J.D.P..
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Table 1. Gene ontology analysis of RNA sequencing data on sorted TAMs from WT and JHU083
treated 4T1 tumor-bearing mice. Molecular functional analysis using gene ontology (GO) in downregulated genes (q value < 0.05)
GO molecular function analysis in downregulated genes in JHU083 treated TAMs Term Count % P-Value
DNA replication 22 0.1 6.7E-13
Cell cycle 42 0.2 7.2E-12
Proteasome 19 0.1 0.00000031
Spliceosome 32 0.2 0.0000015
Pyrimidine metabolism 26 0.1 0.0000072
ECM-receptor interaction 23 0.1 0.000019
Pentose phosphate pathway 11 0.1 0.00014
Aminoacyl-tRNA biosynthesis 14 0.1 0.00018
One carbon pool by folate 8 0 0.00057
Mismatch repair 8 0 0.0049
Base excision repair 11 0.1 0.0058
Glycolysis / Gluconeogenesis 15 0.1 0.0078
Purine metabolism 27 0.2 0.0087
Nucleotide excision repair 11 0.1 0.0099
Terpenoid backbone biosynthesis 6 0 0.01
Small cell lung cancer 17 0.1 0.011
Oocyte meiosis 21 0.1 0.012
Galactose metabolism 8 0 0.016
RNA degradation 13 0.1 0.017
Glyoxylate and dicarboxylate metabolism 6 0 0.018
Steroid biosynthesis 6 0 0.024
Parkinson's disease 22 0.1 0.028
Arginine and proline metabolism 11 0.1 0.04
Table 2. Gene ontology analysis of RNA sequencing data on sorted TAMs from WT and JHU083
treated 4T1 tumor-bearing mice. Molecular functional analysis using gene ontology (GO) in upregulated genes (q value < 0.05)
GO molecular function analysis in upregulated genes in JHU083 treated TAMs
Figure 1. Glutamine antagonism inhibits tumor growth and lung metastasis in an immunedependent manner. (A-F) 1x105 4T1 cells were implanted subcutaneously into the mammary fat pad ofBALB/cJ female mice. On day 7, 10, 13, 17, and 24, mice were injected IP with 250 μg anti-PD1 and/or100 μg anti-CTLA4 antibodies. 4T1 tumor-bearing mice were treated with JHU083 (1mg/kg) starting atday 7 after tumor inoculation. After 7 days of treatment, a lower dose (0.3 mg/kg) of JHU083 was used.(A) Tumor size was monitored (N=5/group). (B) On day 17, tumor weight was measured. (C) Thestructure of the glutamine antagonist prodrug, JHU083. 6-Diazo-5-oxo-L-norleucine (DON) is depictedin black and its ethyl and 2-Amino-4-methylpentanamido promoieties are depicted in blue and red,respectively. (D-F) The whole lungs were harvested, and spontaneous lung metastasis were analyzed. (D-E) To quantify tumor nodules, on day 30, lungs were inflated with 15% india ink. (D) Representativepicture of lungs. (E) Quantification of tumor nodules in lungs (N=16-19/group, three experimentscombined). (F) Representative histology sections stained with H&E. (G) 1x105 4T1 cells were implantedsubcutaneously into the mammary fat pad in WT BALB/cJ, RAG1 KO, or NSG female mice. 4T1 tumor-bearing mice were treated with JHU083 (1mg/kg) daily starting at day 7 after tumor inoculation. After 7days of treatment, a lower dose (0.3 mg/kg) of JHU083 was used. Tumor burden and survival wereassessed (N=5/group). Data are representative of at least three independent experiments. NS: nosignificant. **p<0.001, ****P<0.001, Mean ± S.D. Two way ANOVA with Tukey multiple comparisonspost-test (A), Kruskal-Wallis test with Dunn's multiple comparisons post-test (B), Mann-Whitney tests(E) and Log-rank (Mantel-Cox) test (G).
Cel
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(E)
Figure 2. Glutamine antagonism inhibits infiltration of MDSCs in both primary tumor and lungmetastatic site. 1x105 4T1 cells were implanted subcutaneously into the mammary fat pad of BALB/cJ femalemice. On day 7, 10, 13, 17, and 24, mice were injected IP with 250 μg anti-PD1 and/or 100 μg anti-CTLA4antibodies. 4T1 tumor-bearing mice were treated with JHU083 (1mg/kg) starting at day 7 after tumorinoculation. After 7 days of treatment, a lower dose (0.3 mg/kg) of JHU083 was used (N=5/group). (A) Onday 17, percentages of PMN-MDSCs (CD11b+F4/80negLy6CloLy6Ghi) and Mo-MDSCs(CD11b+F4/80negLy6ChiLy6Gneg) of live cells from the blood were analyzed by flow cytometry (N=5/group).(B) On day 14, tumors were harvested and tumor-infiltrating immune cells were analyzed by flow cytometry.The populations of PMN-MDSCs and Mo-MDSCs were shown. (C) Each cell population numbers werecounted. Total cell numbers were divided by respective tumor weights (mg). (N=5-10/group) were shown. (D)The number of TAMs (CD11b+F4/80+CD8negLy6CnegLy6Gneg) per mg was shown. (E) On day 14, lungs fromsubcutaneously injected 4T1 tumor-bearing mice were harvested. PMN-MDSCs and Mo-MDSCs among lung-infiltrating immune cells were counted. (N=3/group) Data are representative of at least three independentexperiments. NS: no significant. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, Mean ± S.D. One wayANOVA with Tukey multiple comparisons post-test (A, B and E). Mann-Whitney tests (C and D).
4T1 Blood 33hrsMo-MDSCs
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e-3
% n
orm
aliz
ed to
NT *
*
NTJHU083
7hrs
(1dos
e)
33hrs
(2do
ses)
72hrs
(3do
ses)
0
100
200
300
400
500
Activ
e ca
saps
e-3
% n
orm
aliz
ed to
NT NT
JHU083****
***
(B)
Active caspase-3
PMN-MDSCs blood
Mo-MDSCs blood
Active caspase-3
Actin
MDSCs
NT DON1µM
NT JHU0830
1
2
3
4
Cel
l num
bers
(X10
5 ) *
TIL MDSCs7hrs
NT JHU0830.00
0.05
0.10
0.15
Seru
m C
SF3
(O.D
. Va
lue) **
NT JHU0830
2
4
6
8
10
CSF
3 ng
/mL
in 1
50m
g tu
mor
**
In vivo 4T1 tumor lysates
In vivo 4T1 Serum
In vitro 6hrs
NT DON 1µM0.0
0.5
1.0
1.5
Csf
3m
RN
A(re
lativ
e ex
pres
sion
)
*
C/EBPβ
Actin
Figure 3
(C)
(A)
(D)(E)
7hrs
(1dos
e)
33hrs
(2do
ses)
72hrs
(3do
ses)
0
20
40
60
% P
MN
-MD
SCs
of li
ve C
D45
+
****
7hrs
(1dos
e)
33hrs
(2do
ses)
72hrs
(3do
ses)
0
5
10
15
20
% M
o-M
DSC
sof
live
CD
45+ ***NT
JHU083
4T1 PMN-MDSCs 4T1 Mo-MDSCs
DON (μM) 0 1
EV NT
EV JHU08
3
CSF3 OE N
T
CSF3 OE JH
U083
0
5
10
15
20
25
% P
MN
-MD
SCs
of li
ve C
D45
+
***
***
EV NT
EV JHU08
3
CSF3 OE N
T
CSF3 OE JH
U083
0
5
10
15
20
25
% M
o-M
DSC
s of
live
CD
45+
********NS
NT JHU0830.0
0.5
1.0
1.5
Csf
3 m
RN
A(R
elat
ive
expr
essi
on) *
In vivo 4T1 tumor lysates
EV NT
EV JHU08
3
C/EBPβ O
E NT
C/EBPβ O
E JHU08
30
10
20
30
40
% P
MN
-MD
SCs
of li
ve C
D45
+
********
EV NT
EV JHU08
3
C/EBPβ O
E NT
C/EBPβ O
E JHU08
30
5
10
15
20
% M
o-M
DSC
s of
live
CD
45+
*****(F)
(G)
CSF3 OE 4T1 PMN-MDSCs
CSF3 OE 4T1 Mo-MDSCs
C/EBPβ OE 4T1 PMN-MDSCs
C/EBPβ OE 4T1 Mo-MDSCs
In vitro 24hrs
C/EBPβ
NT JHU083
In vivo Sorted GFP+ 4T1
Actin
% M
ax
NT JHU083
Figure 3. Glutamine antagonism reduces MDSCs by increasing cell death and inhibiting tumorCSF3 secretion. (A) MDSCs from 4T1 tumor-bearing mice were treated with DON (1µM) for 24hrs,and active caspase-3 level was analyzed by immunoblot. Actin was used as loading control. (B-D) 4T1tumor-bearing mice were treated with JHU083 (1 mg/kg) starting at day 14 after tumor inoculation. (B)After 7hrs of first treatment and following every day treatment, active caspase-3 on PMN-MDSCs andMo-MDSCs from blood were analyzed by flow cytometry at the indicated time point (N=5/group). (C)Cell numbers and percentages of MDSCs from tumor were counted and analyzed by flow cytometry(N=5/group). (D) Serum (N=16/group) and tumor (N=4/group) were collected from 4T1 tumor-bearing mice and CSF3 was measured by ELISA (top). After 6hrs treatment with or without DON(1µM), Csf3 mRNA levels were measured in 4T1 cells (bottom left) (N=3, technical replication). Csf3mRNA levels were measured from in vivo 4T1 tumor lysates by q-PCR (N=5mice) (bottom right). (E)Percentages of PMN-MDSCs and Mo-MDSCs from empty vector (EV) 4T1 or CSF3 overexpressed(OE) 4T1 tumor bearing mice were analyzed by flow cytometry. (F) C/EBPβ levels were measured onGFP+ sorted tumor cells from 4T1 tumor bearing mice (left) and on 4T1 tumor cells with or withoutDON (1µM) treated for 24hrs (right) by immunoblotting. (G) PMN-MDSCs and Mo-MDSCs from EV4T1 or C/EBPβ OE 4T1 tumor bearing mice were analyzed by flow cytometry. Data are representativeof at least two (E-G) or three (A-D) independent experiments. NS: no significant. *P<0.05, **P < 0.01,***P < 0.005, ****P < 0.001, Mean ± S.D. Two-way ANOVA with post multiple t tests (B, C, E andG). Unpaired t tests (D).
Enrichment plot: GO phagocytic vesicleEnrichment plot: GO pattern recognition
receptor signaling pathway
JHU083NT
0 10 20 30 400
500
1000
1500
Days Post 4T1 injection
Tum
or V
olum
e (m
m3 )
WT JHU083 Isotype abWT JHU083 aTNFa depleting Ab
*
RNA sequencing
MHCII CD86 CD80 iNOSTLR4
7201273
8891264
17762445
875352
938219233
% M
ax
Figure 4. Glutamine antagonism induces reprogramming of TAMs from a suppressive to a pro-inflammatory phenotype. (A) The percentages of TAMs from vehicle or JHU083 treated 4T1 tumor-bearing mice (on day 17). (B) Volcano plot showing significant changes in gene expression (red) fromRNA sequencing analysis on NT and JHU083 treated TAMs(CD11b+F4/80+7AADnegLy6CnegLy6GnegCD8neg) from 4T1 tumor-bearing mice (on day 14). q-value<0.05. (C) Gene set enrichment analysis (GSEA) plot of phagocytic vesicle and pattern recognitionreceptor signaling activity related genes in NT vs. JHU083 on TAMs. Enrichment scores in gene set isshown. (D) Normalized gene expression from RNA sequencing analysis on NT (black) and JHU083(red) treated TAMs from 4T1 tumor-bearing mice (on day 17). All genes are significant (q-value<0.05). (E) Representative histogram and summary graphs of TLR4, MHCII, CD86, CD80 and iNOSexpression on TAMs. (F) TILs were harvested on day 17 from 4T1 tumor-bearing mice treated with orwithout JHU083. Cells were incubated with golgi-plug in the presence or absence of LPS for 9 hoursex-vivo. Percentages of TNF+ cells were analyzed by flow cytometry (left). MFI of TNF from TNF+
cells (right). (G) Correlation of the % TNF+ secreting of TAMs after stimulation with respective totumor weight (left). 4T1 tumor-bearing mice were treated with JHU083 every day andintraperitoneally injected with isotype antibody or 100µg a-TNF antibody (depleting) twice per weekstarting at day 7 after tumor inoculation (right). Data are from one experiment with 5 mice per group(A-D) or from three independent experiment with 5-10 mice per group (E-G). NS: no significant. *P <0.05, ****P<0.001, Mean ± S.D. Mann-Whitney tests (A). Unpaired t test (E). One way ANOVAwith Tukey multiple comparisons post-test (F) and spearman correlation (G).
Figure 5(A)
(B)
NT
JHU08
3 NT
JHU08
30
20
40
60
% T
NF+ o
f CD
45.1
+ TAM
s
****
NT
JHU08
3 NT
JHU08
30
5
10
15
20
25
% T
NF+ o
f CD
45.2
+ TAM
s
***
CD45.1 mice
4T1 tumor inoculation
4T1 tumorinoculation Harvest for analysis
Adoptive transfer MDSCsfrom CD45.1 tumor bearing mice
-21days -7days 0days 7days
Blood MDSCs (>90%) – from CD45.1 mice
CD45.2 recipient mice CD45.2 recipient mice
MHCII on CD45.1+CD45.2+ Endogenous
TAMs(C)
NTJHU0830
2000
4000
6000
8000
10000
MH
CII
(gM
FI)
*
NTJHU0830
2000
4000
6000
8000
CD
86 (g
MFI
) *
NTJHU0830
500
1000
1500
2000
2500
CD
80 (g
MFI
)
**
CD45.2+ MDSC -->TAMs conversion CD86 on CD45.1+CD80 on CD45.1+
+LPS (ex vivo)+LPS (ex vivo)
JHU083 (1mg/kg/day x D0-D6)
Figure 5. Glutamine antagonism induces differentiation of MDSCs from a suppressive to a pro-inflammatory phenotype. Isolated MDSCs in blood from CD45.1 4T1 tumor bearing mice (21 daysafter 4T1 tumor inoculation) were adoptively transferred into CD45.2 4T1 tumor bearing mice (7daysafter 4T1 tumor inoculation). Then, MDSC recipient CD45.2 4T1 tumor bearing mice were treatedwith JHU083 until harvesting tumors on day 7. (A) Schematic of the experiment. (B) Cells wereincubated with golgi-plug in the presence or absence of LPS for 9 hours ex vivo. Percentages of TNF+
CD45.2+ cells (endogenous, left) and TNF+ CD45.1+ cells (adoptively transferred, right) were analyzedby flow cytometry. (C) Summary graph of MHCII, CD86 and CD80 gMFI. Data are from threeindependent experiment with 5-10 mice per group (B, C). *P < 0.05, **P < 0.01, ***P<0.005,****P<0.001, Mean ± S.D. One way ANOVA with Tukey multiple comparisons post-test (B).Unpaired t test (C).
Figure 6
pNF-κB(s536)
Total NF-κB
0 0.5 5 50
Actin
Active caspase-3
DON (μM)
- 0 0.5 1 3LL CM
DON (μM)
(B)
(D)
NT
DON 1µM
DON 5µM
DON 10µM
0
10
20
30
40
% S
urfa
ce C
alre
ticul
in+
of to
tal c
ell
****
Actin
LAMP2
NTJHU0830
500
1000
1500
Surfa
ce c
alre
ticul
in g
MFI
on G
FP+ 4
T1 tu
mor
cel
ls
**
NT
DON 1µM
DON 5µM
DON 10µM
0
50
100
150
200
Cel
lRox
gM
FI
* * **NT
DON 1μM
DON 5μM
DON 10μM
(A) (C)
1.00 1.581.27 1.33
1.00 1.51.24 1.77
1.00 1.821.59 3.16
CellRox
(E)
B16 OVAOT1 CD8+
B16 OVA + BMDM
+/- DON (1 or 5μM) for 18hrs
Removed supernatant(No DON anymore) B16 OVA +
BMDM
eFluor450 (proliferation dye) labeled CD8+ OTI T cells
After 72hrs harvestedfor flow cytometry
NT
DON 1µM
DON 5µM
0
10
20
30
40
% D
ivid
ed c
ells
of C
D8+
****
(F)
Proliferation dye
NTDON 1μMDON 5μM
WT BMDM
MyD88/TRIF DKO BMDM
TFEB KO BMDM
NT
DON 1μM DON 5μM
Proliferation dye
(G)
NT
- + + +
In vitro In vivo
Figure 6. Glutamine antagonism increases immunogenic cell death and antigen presentation toT cells. (A-C) 4T1 tumor cells were cultured with or without DON (0, 0.5 μM, 1 μM, 5 μM, 10 μM,50 μM) for 24 hours. (A) Cells were harvested and stained with CellROX (ROS measurement), andanalyzed by flow cytometry. Representative histogram (left) and summary graph (right). (B) Cellswere lysed and active caspase-3 was analyzed by immunoblot. (C) Cells were stained for calreticulinand analyzed by flow cytometry. Percentages of surface calreticulin+ were shown (left). Summarygraph of surface calreticulin gMFI on GFP+ gated in vivo tumor cells (right). (D) 3LL cells werecultured with or without DON (0.5 μM or 1 μM). After 1 hour of incubation, cells were washed andreplaced with drug-free media. After 24 hours, supernatants were harvested and used as conditionedmedia (CM). BMDMs were cultured in the presence of these conditioned media for 24 hours.Phospho-NF-κB (ser536) and LAMP2 were measured by immunoblot. (E-G) 3x105 BMDMs and5x104 B16-OVA tumor cells were co-cultured with or without DON (1 μM or 5 μM). After 24 hoursof incubation, supernatants were discarded and 3x105 eFluor450-labeled naïve CD8+ T cells fromOTI mice were added. (E) Schematic of the experiment. (F) Representative flow plot (left) andpercentages of divided cells (right) from CD8+ T cells were analyzed by flow cytometry. (G)BMDMs from WT, MyD88/TRIF double KO (DKO) or TFEB KO mice and B16-OVA tumor cellswere co-cultured in the same method as (E), and histogram of the dilution of eFluor450-labeled CD8+
T cells is shown. Data are representative of at least three independent experiments. *P < 0.05, ** P <0.01, **** P < 0.001, Mean ± S.D. One way ANOVA with Tukey multiple comparisons post-test (Aand F). Mann-Whitney tests (C).
Proliferation dye
WT BMDM NT
Batf3 KO BMDM DON 1μM
Batf3 KO BMDM NTWT BMDM DON 1μM
(A)
WT NT
WT JHU08
3
Batf3 K
O NT
Batf3 K
O JHU08
3-100
0
100
200
300
400
% o
f cha
nge
in tu
mor
vol
ume
afte
r tre
atm
ent * ***
WT NT
WT JHU08
3
Batf3 K
O NT
Batf3 K
O JHU08
30
200
400
600
800
*
*
Gra
nzym
e B
(gM
FI)
WT NT
WT JHU08
3
Batf3 K
O NT
Batf3 K
O JHU08
30
2000
4000
6000
8000
Ki67
(gM
FI)
*
**
WT NT
WT JHU08
3
Batf3 K
O NT
Batf3 K
O JHU08
30
1000
2000
3000
4000
CD
44 (g
MFI
) *
**
WT NT
WT JHU08
3
Batf3 K
O NT
Batf3 K
O JHU08
30
500
1000
1500
2000
2500
Tum
or w
eigh
t (m
g) *******Figure 7
(B)
(C)
WT NT
WT JHU08
3
Batf3 K
O NT
Batf3 K
O JHU08
30.00.20.40.6
5
10
% C
D8+ TC
Rβ+ /C
D45
+
*
*
Figure 7. Glutamine antagonism increases tumor antigen cross-presentation to T cells bymacrophage. (A) 3x105 WT or Batf3 KO BMDMs and 5x104 B16-OVA tumor cells were co-cultured in the presence or absence of 1 μM of DON. After 24 hours of incubation,supernatants were discarded and 3x105 eFluor450-labeled naïve CD8+ T cells from OTI micewere added. Histogram of divided cells from CD8+ T cells were analyzed by flow cytometry.(B-C) 5x105 MC38 cells were implanted subcutaneously into flank of WT C57BL/6J mice orBatf3 KO mice. MC38 tumor-bearing mice were treated with JHU083 (0.3 mg/kg) dailystarting at day 14 after tumor inoculation. (B)Tumor weight was recorded (left), and percentchange in tumor volume was calculated (right). (C) On day 21, tumors were harvested, andCD8+ T cells were analyzed by flow cytometry. Data are representative of three independentexperiments. *P < 0.05, **P < 0.01, ***p < 0.005, ****P < 0.001, Mean ± S.D. One wayANOVA with Tukey multiple comparisons post-test (B). Unpaired t tests (C).
NTJHU0830.00
0.05
0.10
0.15
0.20
CD
8:M
DSC
s ra
tio
**
NTJHU0830
5
10
15
20
% C
D8+ o
f liv
e C
D45
+ ***
4T1 TIL
NTJHU0830.00
0.02
0.04
0.06
0.08
CD
8:M
DSC
s ra
tio
*
NTJHU0830
1
2
3
4
5
% C
D8+ o
f liv
e C
D45
+ *Lung
(C)
(A)
(D)
4T1 TIL
Lung
(B)
0 10 20 30 40 500
1000
2000
3000
4000
Days Post 4T1 injection
Tum
or v
olum
e (m
m3 )
0 10 20 30 40 500
1000
2000
3000
4000
Days Post 4T1 injection
Tum
or v
olum
e (m
m3 )
0 10 20 30 40 500
1000
2000
3000
4000
Days Post 4T1 injection
Tum
or v
olum
e (m
m3 )
0 10 20 30 40 500
1000
2000
3000
4000
Days Post 4T1 injection
Tum
or v
olum
e (m
m3 )
NT aPD1+aCTLA4
JHU083 JHU083+aPD1+aCTLA4
0 20 40 600
50
100
Days Post 4T1 Injection
Perc
ent s
urvi
val (
%)
NTaPD1+aCTLA4JHU083JHU083+aPD1+aCTLA4
<0.0001
**
Figure 8
Figure 8. Glutamine antagonism enhances the efficacy of immune-checkpoint blockade inimmunotherapy resistant tumor. (A) Percentages of CD8+ cells and ratio of CD8+ T cells to MDSCsfrom tumor and lung from subcutaneously injected 4T1 tumor bearing mice were analyzed by flowcytometry (N=5-10/group). (B-C) 4T1 tumor-bearing mice were treated with vehicle or JHU083alone, or vehicle or JHU083 in combination with 250 μg anti-PD1 and 100 μg of anti-CTLA4antibodies (On day 7, 10, 13, 17, and 24). (B) Tumor sizes and (C) survival curves were recorded. (D)Proposed model. Data are representative of three independent experiments. *P < 0.05, **P < 0.01,****P < 0.001, Mean ± S.D. Mann-Whitney t tests (A). Log-rank (Mantel-Cox) test (C).
Figure 9. Glutamine antagonism alters metabolism of primary tumor and metastatic sites inglutamine dependent and independent pathways. 1x105 4T1 cells were implantedsubcutaneously into mammary fat pad of BALB/cJ female mice. 4T1 tumor-bearing mice weretreated with JHU083 (1 mg/kg) starting at day 7 after tumor inoculation. After 7 days of treatment,a lower dose (0.3 mg/kg) of JHU083 was used. On day 17, tumors were harvested, and equal massof tumor samples were subjected for LC-MS analysis (A) Principal component (PC) analysisbetween NT (vehicle treated, green) and JHU083-treated (pink) groups. (B) Heatmap visualizationof the metabolite changes between NT (green) and JHU083-treated (red) groups. (C) Volcano plotof metabolites were shown. Log2 fold change vs. –log10 (FDR corrected P value) representingsignificant metabolite changes. Pink: significant. Data are from one experiment with 4-5 mice pergroup. t test (B, C).
Figure 10. Glutamine antagonism alters metabolism of metastatic sites in glutaminedependent and independent pathways. 1x105 4T1 cells were implanted subcutaneously intomammary fat pad of BALB/cJ female mice. 4T1 tumor-bearing mice were treated with JHU083 (1mg/kg) starting at day 7 after tumor inoculation. After 7 days of treatment, a lower dose (0.3mg/kg) of JHU083 was used. On day 17, whole lungs from mice were harvested, and whole lunglysates were subjected for LC-MS analysis. (A) Principal component analysis between the vehicletreated (NT) (green) and JHU083 (pink) treated lungs and (B) heatmap visualization of themetabolite changes between NT (green) and JHU083 (red) treated group were shown. (C) Venndiagram displaying shared significantly changed (1.5 fold, p<0.05) metabolites from tumor andlung. (D) Relative amounts of kynurenine from tumor (left) and lung (right) between NT andJHU083 treated group. Data are from one experiment with 4-5 mice per group. **P < 0.01,****P<0.001, Mean ± S.D. t test (B) and Mann-Whitney tests (D).
NS
DON 1µM
IFNγ
DON 1µM+IF
Nγ0
200
400
600
800
Ido
mR
NA
(rela
tive
expr
essi
on)
****
(F)
(G)
N o rma l
B rea s t t
umo r
P P A T
P F A S
G F A T
N A D S Y N 1
G L U L
C T P S 1
G M P S
G L S 1
A S N S
C A D
0
0 .0 1
Normal
Breast
tumor
CTPS1
GMPS
GLS1
ASNS
CAD
00.000010.000020.000030.000040.00005
TCGA normal or breast tumor dataGlutamine utilizing enzymes
Figure 11. Glutamine antagonism reduces IDO expression by decreasing p-STAT1/3 signaling.(A-D) 1x105 4T1 cells or 1x105 GFP+4T1 cells were implanted subcutaneously into mammary fat pad ofBALB/cJ mice. 5x105 MC38 cells were implanted subcutaneously into flank of C57BL/6 mice. Tumor-bearing mice were treated with JHU083. On day 21, IDO expression in tumor lysates from (A) 4T1 or(B) MC38 tumor-bearing mice was measured by immunoblot. On day 12, (C) GFP+ tumor cells, (D)TAMs and MDSCs were sorted. Cells were lysed and IDO expression was measured by immunoblot.(E) The ratio of kynurenine to tryptophan in tumor (F) 4T1 cells were cultured in the presence orabsence of DON (0.5 μM or 1 μM) and IFNɣ for 6 or 24 hours. p-STAT1 (s727) and IDO expressionwere measured by immunoblot (left). After 6hrs with DON 1 μM treatment, Ido mRNA levels weremeasured by q-PCR (right). (G) RAW264.7 cells were cultured in the presence or absence of DON (0.5μM or 1 μM) and IFNɣ for 6 or 24 hours. p-STAT1 (s727), p-STAT3 (s727) and IDO were measured byimmunoblot (left). After 6 hours with DON (1 μM) treatment, Ido mRNA levels were measured by q-PCR (right). (H) 4T1 tumor-bearing mice were treated with JHU083. On day 14, IDO expression withinlung lysates from tumor free, and 4T1 tumor-bearing mice with or without JHU083 treatment wasmeasured by immunoblotting. (I) Heat map visualization of p-value from pearson correlation analysis(non-log scale for calculation) using TCGA normal and breast invasive carcinoma data between theglutamine utilizing enzymes which are inhibited by DON and IDO expression (left). The graphs fromeach enzyme and IDO correlation data (right). *P<0.05, **P < 0.01, ****P<0.001, Mean ± S.D. Mann-Whitney t tests (E). One way ANOVA with Tukey multiple comparisons post-test (F and G).
Glutamine antagonist
Glutamine sufficient
Killing
Danger signals
TNF
T cells
MDSCs
Tumor cells
IDO
KillingTryp
Kyn
TAMs
TAMs
Tumor cells
CSF3CEBPB
MDSCs
IL-10
IDO
IDO
IDO
IDO
IDO
KynTryp
T cells
Cell type Mechanism
Tumor
① Inhibit Growth Decreased purine, pyrimidine, protein and lipid synthesis
② Decreased MDSC
recruitment
Decreased C/EBPβ leading to decreased CSF3 production
③ Immunogenic Cell death
Increased ROS and release of DAMPs
④ Decreased IDO
Decreased STAT1 mediated transcription
Myeloid Cells
⑤ Increased apoptosis Caspase-3 induced cell death
⑥ Conversion to InflammatoryMacrophages
Increased NF-kB activation
⑦ Enhanced M1 activation
MyD88/TRIF and TFEB dependent activation by DAMPs
①
②
③
④
⑤
⑥
⑦
Figure 12
Figure 12. The proposed mechanisms of how glutamine antagonism enhances anti-tumor immunity. Proposed models describing the effect of glutamine antagonism on the heterogeneous tumor micro-environment.