Figure 8. MNK and mTOR cooperate to regulate T cell differentiation. A) Formation of the immune synapse between T cells and Antigen Presenting Cells (APC) induces mTOR signaling, among other signaling pathways, to stimulate T cell activity. As the T cell divides, a gradient of mTOR signaling radiates from the immune synapse such that the proximal daughter cell exhibits high mTOR activity (blue) while the distal daughter cell exhibits lower mTOR activity (red). Low mTOR activity correlates with the establishment of T Stem Cell Memory (T SCM ) cells, while high mTOR activity promotes differentiation toward Effector T cells. eFT508 inhibits MNK activity and dampens mTOR signaling promoting a T SCM fate (middle). Moderate mTOR inhibition by low concentrations of Rapamycin blocks Effector T cell activation, but allows some level of T SCM generation (right). B) Activated T cells differentiate from T SCM cells, with a high regenerative capacity, toward Effector T cells, which harbor maximal cytotoxic potential. Physical and Functional Interactions between MNK and mTOR Signaling Regulate the Activation and Differentiation of T cells Craig R Stumpf, Vikas K Goel, Rajesh K Sharma, Gary G Chiang, Peggy A Thompson, and Kevin R Webster eFFECTOR Therapeutics, San Diego, CA Abstract The role of the PI3K/mTOR and MAPK signaling pathways in regulating T cell activation and differentiation is well established. It has been shown that mTOR acts as a sensor of the T cell metabolic state, coordinating diverse inputs to determine the balance between effector versus memory cell fates. Similarly, the MAPK interacting kinases, MNK1 and MNK2, are key downstream effector kinases that mediate post-transcriptional gene regulation of critical mediators of the T cell response, including immune checkpoint proteins and cytokines, via the phosphorylation of specific RNA binding proteins such as eIF4E and hnRNPA1. eFT508 is a potent and selective inhibitor of both MNK1 and MNK2, which enhances anti-tumor immune responses by decreasing the expression of immune response modulators including PD-1, PD-L1, LAG3, TIM3 and IL-10. Furthermore, eFT508 treatment boosts memory T cell populations while increasing the effectiveness of cytotoxic T lymphocytes. Phosphoproteomic analysis of the effects of eFT508 on early T cell activation identified novel eFT508 sensitive phosphopeptides, including a subset that overlaps with mTOR-dependent phosphorylation sites. In vitro biochemical analysis has shown that eFT508 is not a mTOR kinase inhibitor and that most of these sites are not direct MNK substrates. These results suggest inhibition of MNK by eFT508 can have significant effects on mTOR signaling. Consistent with these findings, we observe a physical association between MNK and mTOR that is disrupted by either eFT508 or the allosteric mTOR inhibitor rapamycin. Our phosphoproteomic analysis also identified novel MNK-dependent phosphorylation sites within the translation initiation factor eIF4G1 that regulate the ability of mTOR to recognize eIF4G1 as a substrate. In addition, phosphorylation of the mTOR target 4EBP1 is decreased upon treatment of T cells with eFT508, although 4EBP1 itself is not a direct substrate of MNK in vitro. These data are consistent with a role for MNK in regulating mRNA translation, not only through phosphorylation of eIF4E, but also by modulating the activity of mTOR toward specific substrates. Given the role of mTOR in controlling memory T cell differentiation, our results provide a potential mechanistic basis to explain the impact of MNK inhibition on T cell differentiation. Ongoing studies are further characterizing the consequences of eFT508 regulation on MNK/mTOR signaling and T cell differentiation. This work significantly expands our current understanding of eFT508’s mechanism of action as well as its ability to promote a prolonged anti-tumor immune response. Introduction Results Conclusions Figure 3. eFT508 affects the phosphorylation of both MNK and mTOR substrates in addition to dissociating MNK from mTOR complexes. A-B) Primary human T cells were stimulated with a-CD3/CD28 beads in the presence of increasing concentrations of eFT508 (0-10 mM) for 24 hours prior to measuring the levels of p-4EBP S65 and p-eIF4E S209 . C-D) Primary human T cells treated with eFT508 (1 mM), Rapamycin (0.1 mM), or TAK-228 (0.1 mM) prior to TCR stimulation for 24 hours. The phosphorylation of mTOR substrates was monitored by western blot. The quantitation of p-SQSTM1 relative to DMSO treatment is plotted in (D). E) HAP1 cells expressing Flag-MNK2 were treated with eFT508 (1 mM), Rapamycin (0.1 mM), and TAK-228 (0.1 mM) prior to a-Flag-MNK2 immunoprecipitation. eFT508, Rapamycin, and to a lesser extent, TAK-228 decrease the association of mTOR, Raptor, and Rictor with MNK2. • eFT508 disrupts MNK-mTOR complexes and alters the phosphorylation of select mTOR substrates • eFT508 skews T cell differentiation toward T SCM and T CM populations, supporting a prolonged anti-tumor immune response • eFT508 is currently being evaluated in phase 2 clinical trials in combination with approved checkpoint therapies (NCT03616834) and as monotherapy for advanced Castrate-Resistant Prostate Cancer (NCT03690141) 4142 Figure 2. eFT508-sensitive phosphorylation sites overlap with mTORC1 dependent phosphorylation sites. A) 14% of established mTORC1-dependent phosphosites in T cells are sensitive to eFT508. B) Relative in vitro kinase activity of MNK1, MNK2, and mTOR upon treatment with eFT508 illustrates that eFT508 does not directly inhibit mTOR kinase activity. C) MNK2 does not directly phosphorylate the majority of reported mTOR sites in vitro. Figure 1. MNK and mTOR cooperate to integrate TCR signaling and promote T Effector cell differentiation. Antigen recognition by the T Cell Receptor (TCR) combined with activation of costimulatory receptors initiates signaling through multiple pathways leading to T cell activation. mTOR is an established mediator of these signaling networks and is required for T cell activation and T Effector cell differentiation. Complete ablation of mTOR signaling blocks T cell activation and T Effector cell function. eFT508 inhibits MNK and dampens mTOR activity, promoting the development of a T Stem Cell Memory phenotype, which is beneficial for establishing a prolonged anti-tumor immune response. Do Not Post Vinculin p-eIF4E p-S6-240/244 p-eIF4G-1147 p-4EBP-37/46 p-S6-235/236 p-eIF4G-1145 eFT508 Rapamycin - - + - - - - + + - - - TAK-228 mTOR Raptor Rictor MNK2 + - - + - - + - - - - - eFT508 Rapamycin TAK-228 Vinculin p-eIF4E p-eIF4G-1147 p-eIF4G-1145 + - - + - - + - - - - - eFT508 Rapamycin DMNK2 - + + + + - C. B. E. A. B. D. C. A. Percentage of mTOR phosphosites sensitive to eFT508 14% mTOR eFT508 Figure 7. eFT508 promotes the establishment of a T Stem Cell Memory pool. A) Primary human T cells were stimulated with a-CD3/CD28 beads for 24 hours in the presence of the indicated concentrations of eFT508 (green) or Rapamycin (blue). T cells were stained for CD25 as a marker of activation and the percent CD25 + T cells is plotted. B-C) Primary human T cells were stimulated as above in the presence of the indicated concentrations of eFT508. T cells were stained for CD8, CD45RO, and CD27 to identify T Stem Cell Memory (T SCM ) cells. B). The percent of total CD8 + cells that are CD45RO - CD27 + T SCM cells is plotted. C) The percent of T SCM cells that stained positive for Tbet is plotted. A. B. C. eFT508 indirectly decreases mTOR activity via inhibition of MNK B. p-4EBP-S65 Vinculin p-eIF4E eFT508 alters mTOR complex composition and activity A. eFT508 promotes the establishment of a T SCM pool Competitive regulation of translation initiation factors by MNK and mTOR Figure 4. Phosphorylation of adjacent sites on eIF4G by MNK and mTOR are antagonistic toward one another. A) MNK2 phosphorylates eIF4G S1145 (red) in vitro. Phosphorylation of eIF4G S1145 by MNK is inhibited by p-eIF4G S1147 (blue) an mTOR substrate. B) MNK2 phosphorylates eIF4G S1145 in HAP1 cells. Inhibition of MNK by eFT508 blocks phosphorylation of eIF4G S1145 and induces phosphorylation of eIF4G S1147 by mTOR. Figure 6. eFT508 regulates T cell differentiation and enhances the formation of the T Central Memory (T CM ) pool. A) BALB/c peritoneal macrophages were incubated with the indicated concentrations of eFT508 for 1 day prior to mixing with panned splenocytes from C57BL/6 mice in an MLR reaction for an additional 4 days in the presence of the indicated concentrations of eFT508. B) Splenocytes from OT-I mice were stimulated with 5 mg/ml SIINFEKL peptide in the presence of the indicated concentrations of eFT508 for 4 days. In both experiments, cells were analyzed for CD8, CD44 and CD62L expression by flow cytometry. Representative scatter plots for CD44 and CD62L expression in CD8 + cells are shown. CD44 high CD62L low define T Effector Memory cells (T EM ) and CD44 high CD62L high define T Central Memory cells (T CM ). A. B. CD44 CD62L eFT508 promotes the establishment of a T CM pool Figure 5. eFT508 increases cytotoxic T cell function. Splenocytes from OT-I mice were stimulated with SIINFEKL peptide in the presence of the indicated concentrations of eFT508 for 3 days. OT-I splenocytes were washed and mixed at a 10:1 ratio with B6.SJL target splenocytes (1:1 mix of SIINFEKL-pulsed CellTrace high and unpulsed CellTrace low populations) for 16 hours in the absence of eFT508. Target B6.SJL cells were gated by CD45.1 expression and analyzed for CellTrace Violet levels by flow cytometry. The percent cell killing relative to target cells alone is shown in red. Target cells alone Unstim. OT-I SIINFEKL + 0.01 mM eFT508 + 0.1 mM eFT508 + 1 mM eFT508 + 3 mM eFT508 + 10 mM eFT508 11% 49% 52% 53% 64% 73% 81% eFT508 enhances cytotoxic T cell killing A model for T SCM cell enrichment by eFT508-mediated mTOR regulation eFT508 B. MNK does NOT phosphorylate most mTOR substrates in vitro LAT SOS RAS ERK PI3K AKT CD28 ZAP70 GRB2 TCR CD8 mTOR MNK eIF4G eIF4E MNK mTOR Activity Anabolic Metabolism Catabolic Metabolism Glycolysis Fatty Acid Oxidation Differentiation and Expansion Protein Synthesis Autophagy Self Renewal mTOR RAPTOR mTOR RICTOR LCK …VQRSS 1145 LS 1147 RERG… A. T Stem Cell Effector T Cell Untreated T Stem Cell Effector T Cell Rapamycin eFT508 T Stem Cell Effector T Cell mTOR Activity Differentiation & Cytotoxic Potential Self Renewal Priming / Activation Enriched by eFT508 Central Memory T Cell (T CM ) T Stem Cell (T SCM ) Naïve T Cell Effector Memory T Cell (T EM ) Effector T Cell Exhausted T Cell [eFT508], mM [Rapa], mM [eFT508], mM [eFT508], mM
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Figure 8. MNK and mTOR cooperate to regulate T cell differentiation. A) Formation of the immunesynapse between T cells and Antigen Presenting Cells (APC) induces mTOR signaling, among other signalingpathways, to stimulate T cell activity. As the T cell divides, a gradient of mTOR signaling radiates from theimmune synapse such that the proximal daughter cell exhibits high mTOR activity (blue) while the distaldaughter cell exhibits lower mTOR activity (red). Low mTOR activity correlates with the establishment of TStem Cell Memory (TSCM) cells, while high mTOR activity promotes differentiation toward Effector T cells.eFT508 inhibits MNK activity and dampens mTOR signaling promoting a TSCM fate (middle). ModeratemTOR inhibition by low concentrations of Rapamycin blocks Effector T cell activation, but allows some levelof TSCM generation (right). B) Activated T cells differentiate from TSCM cells, with a high regenerative capacity,toward Effector T cells, which harbor maximal cytotoxic potential.
Physical and Functional Interactions between MNK and mTOR Signaling Regulate the Activation and Differentiation of T cellsCraig R Stumpf, Vikas K Goel, Rajesh K Sharma, Gary G Chiang, Peggy A Thompson, and Kevin R WebstereFFECTOR Therapeutics, San Diego, CA
AbstractThe role of the PI3K/mTOR and MAPK signaling pathways in regulating T cell activationand differentiation is well established. It has been shown that mTOR acts as a sensor ofthe T cell metabolic state, coordinating diverse inputs to determine the balance betweeneffector versus memory cell fates. Similarly, the MAPK interacting kinases, MNK1 andMNK2, are key downstream effector kinases that mediate post-transcriptional generegulation of critical mediators of the T cell response, including immune checkpointproteins and cytokines, via the phosphorylation of specific RNA binding proteins such aseIF4E and hnRNPA1. eFT508 is a potent and selective inhibitor of both MNK1 and MNK2,which enhances anti-tumor immune responses by decreasing the expression of immuneresponse modulators including PD-1, PD-L1, LAG3, TIM3 and IL-10. Furthermore, eFT508treatment boosts memory T cell populations while increasing the effectiveness ofcytotoxic T lymphocytes. Phosphoproteomic analysis of the effects of eFT508 on early Tcell activation identified novel eFT508 sensitive phosphopeptides, including a subset thatoverlaps with mTOR-dependent phosphorylation sites. In vitro biochemical analysis hasshown that eFT508 is not a mTOR kinase inhibitor and that most of these sites are notdirect MNK substrates. These results suggest inhibition of MNK by eFT508 can havesignificant effects on mTOR signaling. Consistent with these findings, we observe aphysical association between MNK and mTOR that is disrupted by either eFT508 or theallosteric mTOR inhibitor rapamycin. Our phosphoproteomic analysis also identified novelMNK-dependent phosphorylation sites within the translation initiation factor eIF4G1 thatregulate the ability of mTOR to recognize eIF4G1 as a substrate. In addition,phosphorylation of the mTOR target 4EBP1 is decreased upon treatment of T cells witheFT508, although 4EBP1 itself is not a direct substrate of MNK in vitro. These data areconsistent with a role for MNK in regulating mRNA translation, not only throughphosphorylation of eIF4E, but also by modulating the activity of mTOR toward specificsubstrates. Given the role of mTOR in controlling memory T cell differentiation, our resultsprovide a potential mechanistic basis to explain the impact of MNK inhibition on T celldifferentiation. Ongoing studies are further characterizing the consequences of eFT508regulation on MNK/mTOR signaling and T cell differentiation. This work significantlyexpands our current understanding of eFT508’s mechanism of action as well as its abilityto promote a prolonged anti-tumor immune response.
Introduction
Results
ConclusionsFigure 3. eFT508 affects the phosphorylation of both MNK and mTOR substrates in addition todissociating MNK from mTOR complexes. A-B) Primary human T cells were stimulated with a-CD3/CD28beads in the presence of increasing concentrations of eFT508 (0-10 mM) for 24 hours prior to measuringthe levels of p-4EBPS65 and p-eIF4ES209. C-D) Primary human T cells treated with eFT508 (1 mM), Rapamycin(0.1 mM), or TAK-228 (0.1 mM) prior to TCR stimulation for 24 hours. The phosphorylation of mTORsubstrates was monitored by western blot. The quantitation of p-SQSTM1 relative to DMSO treatment isplotted in (D). E) HAP1 cells expressing Flag-MNK2 were treated with eFT508 (1 mM), Rapamycin (0.1 mM),and TAK-228 (0.1 mM) prior to a-Flag-MNK2 immunoprecipitation. eFT508, Rapamycin, and to a lesserextent, TAK-228 decrease the association of mTOR, Raptor, and Rictor with MNK2.
• eFT508 disrupts MNK-mTOR complexes and alters the phosphorylation of select mTOR substrates
• eFT508 skews T cell differentiation toward TSCM and TCM populations, supporting a prolonged anti-tumor immune response
• eFT508 is currently being evaluated in phase 2 clinical trials in combinationwith approved checkpoint therapies (NCT03616834) and as monotherapy foradvanced Castrate-Resistant Prostate Cancer (NCT03690141)
4142
Figure 2. eFT508-sensitive phosphorylation sites overlap with mTORC1 dependent phosphorylation sites.A) 14% of established mTORC1-dependent phosphosites in T cells are sensitive to eFT508. B) Relative invitro kinase activity of MNK1, MNK2, and mTOR upon treatment with eFT508 illustrates that eFT508 doesnot directly inhibit mTOR kinase activity. C) MNK2 does not directly phosphorylate the majority ofreported mTOR sites in vitro.
Figure 1. MNK and mTOR cooperate to integrate TCR signaling and promote T Effector celldifferentiation. Antigen recognition by the T Cell Receptor (TCR) combined with activation ofcostimulatory receptors initiates signaling through multiple pathways leading to T cell activation. mTORis an established mediator of these signaling networks and is required for T cell activation and T Effectorcell differentiation. Complete ablation of mTOR signaling blocks T cell activation and T Effector cellfunction. eFT508 inhibits MNK and dampens mTOR activity, promoting the development of a T Stem CellMemory phenotype, which is beneficial for establishing a prolonged anti-tumor immune response.
Do Not Post
Vinculin
p-eIF4E
p-S6-240/244
p-eIF4G-1147
p-4EBP-37/46
p-S6-235/236
p-eIF4G-1145
eFT508Rapamycin--
+---
-++ -- -
TAK-228
mTOR
Raptor
Rictor
MNK2
+--+
-- +-
---- eFT508
Rapamycin
TAK-228
Vinculin
p-eIF4E
p-eIF4G-1147
p-eIF4G-1145
+--+
-- +-
---- eFT508
Rapamycin
DMNK2
-
+
+
+
+
-
C.
B.
E.
A.
B.
D.
C.
A.
Percentage of mTOR phosphosites
sensitive to eFT508
14%
mTOR
eFT508
Figure 7. eFT508 promotes the establishment of a T Stem Cell Memory pool. A) Primary human T cellswere stimulated with a-CD3/CD28 beads for 24 hours in the presence of the indicated concentrations ofeFT508 (green) or Rapamycin (blue). T cells were stained for CD25 as a marker of activation and thepercent CD25+ T cells is plotted. B-C) Primary human T cells were stimulated as above in the presence ofthe indicated concentrations of eFT508. T cells were stained for CD8, CD45RO, and CD27 to identify T StemCell Memory (TSCM) cells. B). The percent of total CD8+ cells that are CD45RO-CD27+ TSCM cells is plotted. C)The percent of TSCM cells that stained positive for Tbet is plotted.
A.
B. C.
eFT508 indirectly decreases mTOR activity via inhibition of MNK
B.
p-4EBP-S65
Vinculin
p-eIF4E
eFT508 alters mTOR complex composition and activity
A.
eFT508 promotes the establishment of a TSCM poolCompetitive regulation of translation initiation factors by MNK and mTOR
Figure 4. Phosphorylation of adjacent sites on eIF4G by MNK and mTOR are antagonistic toward oneanother. A) MNK2 phosphorylates eIF4GS1145 (red) in vitro. Phosphorylation of eIF4GS1145 by MNK isinhibited by p-eIF4GS1147 (blue) an mTOR substrate. B) MNK2 phosphorylates eIF4GS1145 in HAP1 cells.Inhibition of MNK by eFT508 blocks phosphorylation of eIF4GS1145 and induces phosphorylation ofeIF4GS1147 by mTOR.
Figure 6. eFT508 regulates T cell differentiation and enhances the formation of the T CentralMemory (TCM) pool. A) BALB/c peritoneal macrophages were incubated with the indicatedconcentrations of eFT508 for 1 day prior to mixing with panned splenocytes from C57BL/6 micein an MLR reaction for an additional 4 days in the presence of the indicated concentrations ofeFT508. B) Splenocytes from OT-I mice were stimulated with 5 mg/ml SIINFEKL peptide in thepresence of the indicated concentrations of eFT508 for 4 days. In both experiments, cells wereanalyzed for CD8, CD44 and CD62L expression by flow cytometry. Representative scatter plots forCD44 and CD62L expression in CD8+ cells are shown. CD44highCD62Llow define T Effector Memorycells (TEM) and CD44highCD62Lhigh define T Central Memory cells (TCM).
A.
B.C
D4
4
CD62L
eFT508 promotes the establishment of a TCM pool
Figure 5. eFT508 increases cytotoxic T cell function. Splenocytes from OT-I mice were stimulatedwith SIINFEKL peptide in the presence of the indicated concentrations of eFT508 for 3 days. OT-Isplenocytes were washed and mixed at a 10:1 ratio with B6.SJL target splenocytes (1:1 mix ofSIINFEKL-pulsed CellTracehigh and unpulsed CellTracelow populations) for 16 hours in the absence ofeFT508. Target B6.SJL cells were gated by CD45.1 expression and analyzed for CellTrace Violet levelsby flow cytometry. The percent cell killing relative to target cells alone is shown in red.
Target cells alone Unstim. OT-I SIINFEKL + 0.01 mM eFT508
+ 0.1 mM eFT508 + 1 mM eFT508 + 3 mM eFT508 + 10 mM eFT508
11% 49% 52%
53% 64% 73% 81%
eFT508 enhances cytotoxic T cell killing
A model for TSCM cell enrichment by eFT508-mediated mTOR regulation
eFT508
B.
MNK does NOT phosphorylate most mTOR substrates in vitro
LAT
SOS
RAS
ERK
PI3K
AKT
CD
28
ZAP70 GRB2
TCR
CD
8
mTOR MNK
eIF4G
eIF4EMNK
mTORActivity
Anabolic Metabolism
Catabolic Metabolism
GlycolysisFatty Acid Oxidation
Differentiation and Expansion
Protein Synthesis
Autophagy
Self Renewal
mTOR
RAPTOR
mTOR
RICTOR
LCK
…VQRSS1145LS1147RERG…
A.
T Stem Cell Effector T Cell
Untreated
T Stem Cell Effector T Cell
RapamycineFT508
T Stem Cell Effector T Cell
mTORActivity
Differentiation & Cytotoxic Potential
Self Renewal
Priming /Activation
Enriched by eFT508
Central Memory T Cell (TCM)
T Stem Cell (TSCM)
NaïveT Cell
Effector Memory T Cell (TEM)
Effector T Cell
Exhausted T Cell
[eFT508], mM [Rapa], mM
[eFT508], mM [eFT508], mM
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