eFT508, a Potent and Selective Mitogen-Activated Protein Kinase Interacting Kinase (MNK) 1 and 2 Inhibitor, is Efficacious in Preclinical Models of Diffuse Large B-Cell Lymphoma (DLBCL)Kevin R Webster1, Vikas K Goel1, Ivy NJ Hung1, Gregory S Parker1, Jocelyn Staunton1, Melissa Neal1, Jolene Molter1, Gary G Chiang1, Katti A Jessen1,2, Christopher J Wegerski1, Samuel Sperry1, Vera Huang1, Joan Chen1, Peggy A Thompson1, James R Appleman1,3, Justin T. Ernst1, Stephen E Webber1,4, Paul A Sprengeler1 and Siegfried H Reich1

1eFFECTOR Therapeutics, San Diego, CA; Present address: 2Pfizer, San Diego, CA; 3Annai Systems, Carlsbad, CA; 4Polaris Pharmaceuticals, San Diego, CA

AbstractDysregulated translation of messenger RNA (mRNA) plays a role in the pathogenesis ofmultiple solid tumors and hematological malignancies. MNK1 and MNK2 integrate signalsfrom several oncogenic and immune signaling pathways, including RAS, p38, and Toll-likereceptor (TLR) pathways, by phosphorylating eukaryotic initiation factor 4E (eIF4E) andother key effector proteins including hnRNPA1 and PSF. Through phosphorylation of theseregulatory proteins MNK1 and MNK2 selectively regulate the stability and translation of asubset of cellular mRNA. eFT508 is a potent, highly selective, and orally bioavailable MNK1and MNK2 inhibitor. eFT508 has a half-maximal inhibitory concentration (IC50) of 1-2 nMagainst both MNK isoforms in enzyme assays and inhibits the kinase through a reversible,ATP-competitive mechanism of action. Treatment of tumor cell lines with eFT508 led to adose-dependent reduction in eIF4E phosphorylation at serine 209 (IC50 = 2-16 nM),consistent with previous findings that phosphorylation of this site is solely dependent uponMNK1/MNK2. In a panel of ~50 hematological cancers, eFT508 showed anti-proliferativeactivity against multiple DLBCL cell lines. Sensitivity to eFT508 in TMD8, OCI-Ly3 and HBL1DLBCL cell lines was associated with dose-dependent decreases in production of pro-inflammatory cytokines including TNFα, IL-6, IL-10 and CXCL10. Further evaluation ofeFT508 mechanism of action demonstrated that decreased TNFα production correlatedwith a 2-fold decrease in TNFα mRNA half-life. These findings are consistent with MNK1phosphorylation of specific RNA-binding proteins, e.g. hnRNPA1, that regulate the stabilityand translation of mRNA containing specific AU-rich elements (ARE) in their 3’-untranslatedregions (UTR). Pro-inflammatory cytokines are drivers of key hallmarks of cancer includingtumor cell survival, migration and invasion, angiogenesis, and immune evasion, while alsodriving drug resistance. Therefore, eFT508 was tested in vivo in 7 subcutaneous humanlymphoma xenograft models. Significant anti-tumor activity was observed in the TMD8and HBL-1 ABC-DLBCL models, both of which harbor activating MyD88 mutations. Inaddition, eFT508 combined effectively with components of R-CHOP and with noveltargeted agents, including ibrutinib and venetoclax, in human lymphoma models. Theseresults underscore the potential of eFT508 for the treatment of DLBCL. eFT508 has alsobeen characterized in nonclinical safety pharmacology and toxicology studies. Clinical trialsin patients with hematological and other malignancies are planned.

Introduction

Results

Conclusions

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TMD8 DLBCL - ABC 9.7SU-DHL-2 DLBCL - ABC 4.3HBL1 DLBCL - ABC 7.7OCI-Ly3 DLBCL - ABC 8.0RI1 DLBCL - ABC 15.7SU-DHL-6 DLBCL - GCB 2.3SU-DHL-10 DLBCL - GCB 2.3OCI-Ly7 DLBCL - GCB 1.4Pfeiffer DLBCL - GCB 11.4Raji Burkitt's 11.6AMO1 MM 16.2MV-4-11 AML 2.0

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Figure 1. eFT508 is a potent and highlyselective inhibitor of MNK1 and MNK2kinases. The IC50 of eFT508 wasdetermined against MNK1 and MNK2 by invitro kinase assays. eFT508 was alsoprofiled at 1 µM against 414 kinases usingthe Invitrogen SelectScreen kinaseprofiling service. The IC50 of eFT508 wasdetermined against the hits with >50%inhibition from this screen, DRAK1 andCLK4, by LanthaScreen kinase bindingassay. n.d., not determined

Figure 2. eFT508 inhibits key MNK1/2-dependent signaling pathways in cells. A) TMD8 cells weretreated with the indicated concentrations of eFT508 for 24 h. Cell lysates were subjected to m7-GTPSepharose pull-down and bound proteins were analyzed by immunoblotting with the indicatedantibodies. B) TMD8 cells were treated with the indicated concentrations of eFT508 for 24 h. Cell lysateswere analyzed by immunoblotting with the indicated antibodies.

Figure 3. eFT508 is a potent inhibitor of phospho-eIF4E in hematological tumor cell lines and inhibitsthe growth of select tumor models. A) eFT508 IC50 values were determined for eIF4E S209phosphorylation in the indicated cell lines. Cells were treated with eFT508 for 2 h and cell lysates wereanalyzed for eIF4E phosphorylation by HTRF assay. B) eFT508 IC50 values were determined for cellproliferation in a panel of hematological cell lines. Cells were treated with eFT508 for 72 h and cellproliferation was assessed by CellTiter Glo. ALCL, anaplastic large cell lymphoma; AML, acute myeloidleukemia; B-ALL, B-cell acute lymphoblastic leukemia; B-PLL, B-cell prolymphocytic leukemia; Burkitt’s,Burkitt’s lymphoma; CML, chronic myelogenous leukemia; DLBCL, diffuse large B-cell lymphoma; MCL,mantle cell lymphoma; MM, multiple myeloma; T-ALL, T-cell acute lymphoblastic leukemia

Figure 4. Key MNK-dependenttranslational processes are down-regulated by eFT508. TMD8 cellswere treated with DMSO or eFT508(10 µM, 48 h). Ribosomal profilingidentified ~125 genes that showeddecreased translation rate witheFT508 treatment relative to DMSOtreatment (Log2 fold decrease ≥ -0.75, p-value ≤ 0.01) and GeneOntology biological classificationwas used to categorize the MNK-sensitive genes.

Figure 5. eFT508 inhibits the production of pro-inflammatory cytokines important for tumorigenesis.A) TMD8 cells were treated with the indicated concentrations of eFT508 for 48 h. Cell supernatantswere collected and the indicated cytokines were quantitated by ELISA. B) TMD8 cells were treated with10 µM eFT508 for 24 h. Actinomycin D was added and RNA was harvested from the cells at various timepoints (0-360 min). Quantitation of RNA was performed by TaqMan assay and calculated mRNA half-lives are shown.

Figure 6. eFT508 inhibits the growth of MyD88-mutant DLBCL xenografts in vivo. A) TMD8 xenograft-bearing animals were treated with the indicated dose/schedule/route of eFT508 for 14 days. Left panel,tumor volume measurements; Right panel, body weight measurements taken throughout the tumorstudy. B) TMD8 xenograft-bearing animals were treated with the indicated dose of eFT508 and tumorand plasma samples were harvested at time points (0.5-24 h) post-dose. eIF4E phosphorylation wasmeasured by immunoblot and % inhibition was plotted as a function of the corresponding eFT508plasma concentration. Upper dashed line, EC90 inhibition; Lower dashed line, EC50 inhibition. C) Table oftumor growth inhibition values at day 14 in lymphoma xenograft models for the indicated doses ofeFT508 (QD PO). P-values (in parentheses) were calculated from the mean tumor volume relative tovehicle control using two-way ANOVA

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Figure 8. Rational combination of eFT508 with the targeted agents, ibrutinib or venetoclax isefficacious in vitro and in vivo. A) Combination index (CI) plot of TMD8 cells treated with eFT508 andibrutinib or venetoclax in vitro. CI values were determined at ED25-ED90 levels from cell proliferationdata (CellTiter Glo, 72 h), where CI <1 indicates synergy, CI=1 indicates additivity, and CI >1 indicatesantagonism. B) In vivo single agent and combined activity of eFT508 with ibrutinib in TMD8 xenograftsC) In vivo single agent and combined activity of eFT508 with venetoclax in TMD8 xenografts.

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Figure 7. eFT508 blocks everolimus-induced eIF4E feedbackphosphorylation and is synergistic in combination. A) TMD8 cellswere incubated with DMSO or 20 nM everolimus for 18 h, thentreated for an additional 2 h with the indicated concentrations ofeFT508. Cell lysates were immunoblotted with the indicatedantibodies. B) Combination index (CI) plot of TMD8 cells treatedwith eFT508 and everolimus in vitro. CI values were determined atED25-ED90 levels from cell proliferation data (CellTiter Glo, 72 h),where CI <1 indicates synergy, CI=1 indicates additivity, and CI >1indicates antagonism. C) TMD8 xenograft-bearing animals weretreated with the indicated dose/schedule/route of compounds forthe denoted time period. Tumor volumes were measured andplotted as a function of time.

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Kinase eFT508IC50, nM

% inhibition @1 µM eFT508

MNK1 2.4 100%

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STK17A/DRAK1 131 82%

CLK4 787 60%Remaining 410protein kinases n.d. < 40%

• MNK1 and MNK2 are serine/threonine kinases that integrate signals from several oncogenic and immune signaling pathways such as RAS, p38 and Toll-like receptor pathways

• The phosphorylation of key MNK substrates, such as eIF4E andhnRNPA1, selectively modulate oncogenic protein expressionthrough the regulation of translation initiation and mRNA stability.

• eFFECTOR Therapeutics has discovered eFT508, a potent, small molecule inhibitor of MNK1 and MNK2 activity

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Xenograft Tumor type Genomic markers% Tumor growth inhibition (p-value)

1 mg/kg eFT508 10 mg/kg eFT508TMD8 DLBCL - ABC CD79, MYD88 82% (<0.0001) 76% (<0.0001)

HBL1 DLBCL - ABC CD79, MYD88 66% (0.009) 96% (<0.0001)

RI1 DLBCL - ABC REL, BCL2, MALT1, MYC 5% (>0.1) -33% (>0.1)

WSU-DLCL-2 DLBCL - GCB BCL2, EZH2, FLT3, IRS1, NF1, TP53 23% (>0.1) 31% (>0.1)

SU-DHL-6 DLBCL - GCB BCL2, EGFR, EZH2, PIK3R1, TP53, TSC2 23% (>0.1) 18% (>0.1)

SU-DHL-10 DLBCL - GCB ATM, CDK4, EZH2, FOXO3, MYC, NOTCH1, PTEN, TP53 49% (0.0545) 60% (0.0153)

Ramos Burkitt’s MYC, IL2, PDGFRB, TP53 39% (<0.0001) 25% (0.0017)

C. • eFT508 is a potent, highly selective, orally bioavailable inhibitor of MNK1 and MNK2 kinases

• eFT508 blocks the production of pro-inflammatory cytokines involved in oncogenic processes

• eFT508 is well-tolerated and shows efficacy against MyD88-mutant DLBCL models in vivo

• eFT508 combines effectively with targeted agents and standard ofcare agents (e.g. R-CHOP) in vivo

• Clinical trials in patients with hematological and other malignancies are planned

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eFT508 Regulon~125 genes (2 - 9 fold decrease in translation rate)

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GeneHalf life (min)

DMSO eFT50810 µM

TNFα 48.1 ± 1.7 24.4 ± 13.6

IL-6 34.9 ± 5.2 17.6 ± 7.0

CXCL-10 213.2 ± 6.3 149.7 ± 5.6

IL-8 52.4 ± 2.0 33.7 ± 0.1

IL-10 50.9 ± 14.1 16.6 ± 9.2

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KRW, VKG, INJH, GSP, JS, MN, JM, GGC, KAJ, CJW, SS, VH, JC, PAT, JRA, JTE, SEW, PAS and SHR areemployees and/or shareholders in eFFECTOR Therapeutics. KAJ is currently an employee of Pfizer. JRAis currently an employee of Annai Systems. SEW is currently an employee of Polaris Pharmaceuticals.

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