F018659 Targeting the Stress Response Kinase ... 8. Conclusions •RAPT Therapeutics is...

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Transcript of F018659 Targeting the Stress Response Kinase ... 8. Conclusions •RAPT Therapeutics is...

  • 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 -

    490 4.5 >50000 390 440 16 >25000 22%

    GCN2i -

    282 7.6 >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 Phosphorylation 5. 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 cells Anti-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

    2.0 2.0

    2.1

    2.2

    2.3

    2.4

    C H

    O P

    (f o

    ld c

    h a n

    g e )

    0.00

    0.02

    0.04

    0.5

    1.0

    1.5

    2.0

    G C

    N 2

    (f o

    ld c

    h a n

    g e )

    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)

    RPMI D)

    Actin

    P-GCN2

    Total GCN2

    P-eIF2α

    Total -eIF2α

    6hr low TRP

    GCN2

    CD8 T

    cell

    Decreased Survival and Tumor Killing

    Increased Tumor

    Survival

    Increased Immune

    Suppression

    MDSC

    ↓ Tryptophan↓ Arginine

    ↓ Asparagine ↓ Glucose

    Experimental Design

    CT26 Tumor Model RENCA Tumor Model

    A)

    B)

    D a y 0

    D a y 3

    D a y 6

    0

    2

    4

    6

    8

    1 0

    N o

    : c

    e ll

    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