f018659 targeting the stress response kinase …...8. conclusions •rapt therapeutics is developing...
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8. Conclusions
• RAPT Therapeutics is developing potent and selective
inhibitors of the stress response kinase GCN2
• GCN2i inhibited phosphorylation of GCN2 and EIF2α in
human CD8+ T cells and human MDSC cultured under amino-
acid starved conditions
• Inhibition of GCN2 increased human and mouse CD8+ T cell
proliferation and effector functions when cultured under
amino-acid deprived conditions
• GCN2i reversed both human and mouse tumor-derived
MDSC-mediated suppression and effector functions of CD8+
T cells
• Treatment of human CD33+ MDSC alone with RPT-GCN2i,
reverses the suppressive function of MDSC on CD8+ T cell
• RPT-GCN2i demonstrates moderate single-agent antitumor
effect in CT26 and RENCA mouse prophylactic tumor models
• We are currently investigating various combination treatments
• Thus our data collectively demonstrates that GCN2 is a
promising therapeutic target for the treatment of cancer
Lisa Marshall, Buvana Ravishankar, Deepika Kaveri, Lavanya Adusumilli, Deepa Pookot, Thant Zaw, Raashi Sreenivasan, Mikhail Zibinsky, Jeffrey Jackson, Grant Shibuya, Paul Leger, Omar Robles, Anqi Ma, Andrew Ng, Anton
Shakhmin, Scott Jacobson, Steve Wong, Jerick Sanchez, Justy Guagua, Martin Brovarney, Angela Wadsworth, Delia Bradford, Christophe Colas, Oezcan Talay, George Katibah, Gene Cutler, David Wustrow, Paul Kassner, Dirk
Brockstedt, RAPT Therapeutics, Inc. South San Francisco, CA
The tumor microenvironment (TME) is characterized by deficiencies in oxygen and key
nutrients, such as glucose and amino acids, resulting in an overall immune suppressive
environment. Stromal cells and myeloid-derived suppressor cells (MDSC) within the
tumor create a nutrient-poor environment that inhibits immune function and supports
tumor growth. GCN2 (general control nonderepressible 2), a stress response kinase,
plays a key role in sensing and modulating the response to nutrient deprivation. GCN2
activation in T cells leads to an induction of the integrated stress response pathway and
subsequently to T cell anergy and apoptosis, enhanced MDSC-dependent immune
suppression and tumor growth survival.
Treatment of these nutrient-deprived T cells with an inhibitor of GCN2 (GCN2i) resulted
in rescue of CD4+ and CD8+ T cell proliferation and effector functions as measured by
flow cytometry. In addition, GCN2 inhibition in MDSC alone fully reversed CD33+ MDSC-
induced T cell suppression and effector functions. Using the CT26 colorectal syngeneic
mouse tumor model we demonstrated that the pharmacologic inhibition of GCN2 in-vivo
leads to an observed anti-tumor effect. Furthermore, GCN2 inhibition induced enhanced
tumor specific CD8+ T cell immunity. Our GCN2i is currently being evaluated to further
elucidate the immune contribution in the tumor microenvironment.
The GCN2 pathway is activated in immune and tumor cells during nutrient deprivation,
resulting in functional suppression of the immune response. Our results demonstrate
that inhibition of GCN2 is an attractive approach for relieving T cell suppression and
promoting anti-tumor activity, demonstrating GCN2 as a promising therapeutic target for
the treatment of cancer.
1. Abstract and Introduction
6. GCN2i Inhibits CT26 Growth in Low Amino Acid Conditions
Section Title – Double Box – Font Size = 50
7. GCN2i as a Single Agent Induces Modest Tumor
Growth Inhibition in Multiple Models
© RAPT Therapeutics , Inc. All right reserved.
ID
GCN2
Enzyme
lC50 (nM)
PERK
Enzyme
IC50 (nM)
PKR
Enzyme
IC50 (nM)
HRI
Enzyme
IC50 (nM)
SKOV3
Cellular
pEIF2a
IC50 (nM)
SKOV3
Cellular
Toxicity
IC50 (nM)
Bioavailability
(Rodent %F)
GCN2i -
4904.5 >50000 390 440 16 >25000 22%
GCN2i -
2827.6 >50000 4400 >25000 24 >25000 80%
Cellular
A) Enzymatic and cellular potency for GCN2i-282; B) Potency and selectivity parameters for GCN2i-490 and
GCN2i-282. For enzymatic assays, compounds were incubated with recombinant human kinases and EIF2α-GFP
substrate. Phosphorylation of EIF2α was measured by TR-FRET and used to calculate inhibition of kinase activity.
For cell-based pEIF2α assay, SKOV-3 cells were incubated with compounds and then stimulated with halofuginone
(1 hour) to activate GCN2 and then pEIF2α was measured by AlphaLisa. For toxicity assessment, SKOV3 cells
were incubated with compounds for 72 hours and viability was assessed with CellTiter-Glo reagent.
2. GCN2i Potently Reduces EIF2α Phosphorylation5. Treatment Of Human CD33+ MDSC With GCN2i Reverses Their
Immuno-suppressive Function
A) GCN2 is a stress response kinase detecting
amino acid starvation in the tumor
microenvironment. B) Activation of GCN2 leads to
1) T cell anergy, apoptosis and enhanced Treg
suppression and a decrease overall T cell function
2) increases myeloid-derived suppressor cell
function and 3) increased tumor survival.
3. GCN2 Activation Leads to
Enhanced Tumor Survival Through
the ISR Pathway
A)
Trp starvation
24hrs
GCN2i
96 hrs
Assess T cell proliferation by dye
dilution and CD8 function by IFN-γ and
GZMb expression
T cellsAnti-CD3/CD28 beads + rhIL2
C)B)
D)
4. GCN2i Restores Human T Cell
Proliferation And Function In
Tryptophan Limited Conditions
A) Experimental schematic
B) Western blot analysis of whole cell lysates from
activated T cells starved for 24hrs with the GCN2i
showed decrease in pGCN2, pEIF2alpha
C) CD8+ T cell proliferation was assessed by dye
dilution (CD4 proliferation is similarly increased –
data not shown)
D) T cell functional markers were analyzed by flow
cytometry.
A) CD33+ myeloid cells from healthy donor (isolated and expanded as described) were pre-starved in tryptophan-limited media and pretreated with GCN2i-
490 for 6hrs and then washed. Pre-starved and pretreated MDSC were co-cultured in complete media with activated CD8+ T cells. B) Proliferation (n=4)
and C) IFN𝛾 (n=2) were measured by flow cytometry. Similar increases in CD107a+CD8+ cells were also observed (data not shown). D) Western blot
indicating pathway is activated at 6 hours under low tryptophan condition and GCN2i-490 leads to a decrease in pGCN2.
B)
BALB/c
mice
Day 1Day 0
CT26 or RENCA
tumor cells
(1x105 cells)Compound dosing
0.00
0.05
0.10
0.15
0.20
0.5
1.0
1.5
2.02.0
2.1
2.2
2.3
2.4
CH
OP
(fo
ld c
han
ge)
0.00
0.02
0.04
0.5
1.0
1.5
2.0
GC
N2
(fo
ld c
han
ge)
Vehicle (1% HPMC)
F019282-09 15mpk
F018659
Targeting the Stress Response Kinase GCN2 to Restore Immunity
And Decrease Tumor Cell Survival
In Vivo GCN2 Target Engagement
Enzymatic
A) B)
C) D)
A) C)
GCN2i-490
CT26 cells cultured with vehicle (DMSO),
Asparaginase (ASNase), or GCN2i alone, or
the combination of ASNase and GCN2i. Cell
number was determined after three and six
days. Asparagine depletion by ASNase or
GCN2i has minimal effects on cell growth
alone, but in combination results in a
dramatic reduction in cell number
GCN2i-490
A) Experimental schematic
B) CT26 Tumors from treated or untreated animals were harvested on day 12 and evaluated for levels of GCN2 or CHOP mRNA by qRT-
PCR. GCN2i-282 resulted reduction of GCN2 and CHOP mRNA.
C) Twice daily dosing (BID) of GCN2i-282 results in modest inhibition of CT26 tumor growth compared to vehicle. Minimal effect on body
weight is observed (inset graph).
D) Reduction in growth of RENCA tumors is observed with twice daily dosing of GCN2i-282 (compared to vehicle)
Vehicle (1% HPMC)
GCN2i-282 15mpk BID
A)
B)
RPMID)
Actin
P-GCN2
Total GCN2
P-eIF2α
Total -eIF2α
6hr low TRP
GCN2
CD8 T
cell
Decreased Survival and Tumor Killing
Increased Tumor
Survival
IncreasedImmune
Suppression
MDSC
↓ Tryptophan↓ Arginine
↓ Asparagine ↓ Glucose
Experimental Design
CT26 Tumor Model RENCA Tumor Model
A)
B)
Day0
Day3
Day6
0
2
4
6
8
1 0
No
: c
ell
s (
x 1
0^
6) D M S O
A S N a se
G C N 2 i-4 9 0 - 2 .5 u M
A S N a s e + G C N 2 i-4 9 0
- 2 .5 u M
GCN2i-490
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