Preclinical Evaluation of eFT226, a Novel, Potent and Selective eIF4A Inhibitor with Anti-tumor #1530 Activity in B-cell Malignancies Peggy A. Thompson, Boreth Eam, Nathan P. Young, Sarah Fish, Joan Chen, Maria Barrera, Haleigh Howard, Ana Parra, Jolene Molter, Jocelyn Staunton, Ivy NJ. Hung, Gregory S. Parker, Gary G. Chiang, Christopher J. Wegerski, Andres Nevarez, Jeff Clarine, Samuel Sperry, Alan Xiang, Chinh Tran, Christian Nilewski, Garrick K. Packard, Theodore Michels, Paul A. Sprengeler, Justin T. Ernst, Siegfried H. Reich, Kevin R. Webster eFFECTOR Therapeutics, San Diego, CA Abstract Results Translational control of oncoprotein expression has been implicated in the pathogenesis of eFT226 Selectively Binds to Free eIF4A Inhibition of Key Oncogenes by eFT226 is Mediated Through the 5’-UTR In vivo Tumor Growth Inhibition by eFT226 multiple solid tumors and hematological malignancies. synthesis is tightly regulated largely at the initiation stage through the eukaryotic 4F (eIF4F) A. B. A. B. A. Tumor Growth Body Weight

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B-cell malignancies are often associated with dysregulation of oncogenes or anti-apoptotic u T 0 16 (e.g., c-MYC, CCND1/3, BCL2 and MCL-1) that contain structured 5’-UTRs and 0 5 10 15 0 5 10 15 require enhanced eIF4A activity for translation. Days of TX Days of TX Figure 4. Protein expression of key oncogenes is regulated by eFT226 through the 5’-UTR. (Dosed on Day 1, 8, & 15) (Dosed on Day 1, 8, & 15) eFT226 is a potent and selective eIF4A inhibitor that promotes eIF4A binding to specific PDB ID: 2G9N Doxycycline-inducible constructs A) MYC [ORF only] or MYC [5’-UTR + ORF] and B) MCL1 [ORF only] or Vehicle eFT226 0.2mg/kg eFT226 0.001mg/kg eFT226 1mg/kg mRNA sequences and interferes with the assembly of the eIF4F initiation complex. eFT226 MCL1 [5’-UTR + ORF] were over expressed in the HEK293T cell line. Cells were treated with eFT226 for 3 Figure 1. eFT226 forms a ternary complex with free eIF4A1. A) The eIF4A F163L binding site mutation hr. MYC, MCL1, CCND1 and GAPDH protein levels were analyzed by western blot analysis. Sequence B. selectively inhibits translation of mRNAs containing longer 5’-UTRs, an increased frequency was introduced using CRISPR gene editing. B) Pulldown experiments using biotinylated eFT226 shows % Tumor growth analysis of the 5’-UTR identified polypurine motifs similar to the high affinity binding sites identified by Xenograft Tumor type Genomic markers inhibition (p-value) of uORFs (upstream open ), and polypurine and/or G-quadraplex recognition selective binding to free eIF4A1 not the eFI4F complex. eIF4A1, 4E, 4B and 4B protein levels were wt direct binding providing potential interaction sites for eFT226-induced ternary complex formation and [1mg/kg eFT226] motifs. The sequence dependency of eFT226 translational inhibition was evaluated in an in  analyzed by western blot. 200 M unlabeled eFT226 was used to compete with the biotinylated-eFT226 translational regulation. TMD8 DLBCL - ABC CD79, MYD88 97% (<0.0001) vitro translation assay demonstrating potent inhibition of reporter constructs containing a binding. The eIF4A binding site mutation blocks interaction of eFT226 with eIF4A. HBL1 DLBCL - ABC CD79, MYD88 87% (<0.0001) polypurine motif in the 5’-UTR (IC ~2 nM). Direct binding studies also confirmed the 50 Pfeiffer DLBCL - GCB BCL2 Translocation, STAT3, EZH2 70% (0.0001) formation of a stable ternary complex between eFT226, eIF4A and mRNA oligonucleotides eFT226 is a Sequence Specific Translational Regulator eFT226 Inhibits Growth and Induces Apoptosis in Tumor Cell Lines SU-DHL-6 DLBCL - GCB BCL2 Translocation, EGFR, EZH2, TP53 83% (0.0002) containing polypurine motifs. This results in eFT226 selectively and translationally MYC and BCL2 Translocations, SU-DHL-10 DLBCL - GCB 37% (0.06) downregulating a subset of genes that are important for tumor growth and survival. 2000 EZH2, PTEN, TP53 A. Binding to AGAGAG RNA surface RNA Sequence KD (M) A. eFT226 B. Cell line Tumor type Apoptosis Ramos Burkitt’s MYC Translocation, IL2, PDGFRB, TP53 75% (<0.0001) eFT226 showed potent anti-proliferative activity (GI50 < 15 nM) and an induction of ) GI50, nM

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R ( apoptosis against a panel of B-cell lymphoma cell lines. Sensitivity to eFT226 was TMD8 DLBCL - ABC 4.1 +++ e AGAGAG 8.0 ±0.9 s 1000 Figure 7. eFT226 inhibits the growth of B-cell lymphoma xenografts in vivo. A) TMD8 xenograft-bearing associated with dose-dependent decreases in the oncogenic drivers c-MYC, CCND1/3, BCL2 n SU-DHL-2 DLBCL - ABC 3 +++ o GGCGGC + eFT226 3.19 ±0.03 p animals were treated with the indicated dose of eFT226 Q1W for 15 days. Left panel, tumor volume s HBL1 DLBCL - ABC 5.6 ++ or MCL-1 in ABC (activated B-Cell) and GCB (germinal center B-cell) DLBCL (Diffuse Large B- e 500 GGCGGC 8.0 ±0.3 R eIF4A + eFT226 Pfeiffer DLBCL - ABC 3.7 +++ measurements; Right panel, body weight measurements taken throughout the tumor study. B) Table of eIF4A only cell Lymphoma) subtypes and Burkitt’s lymphoma. eFT226 has good pharmacokinetic CCGCCG + eFT226 9.6 ±0.4 SU-DHL-6 DLBCL - GCB 5.3 + tumor growth inhibition values at day 14-22 in lymphoma xenograft models for the indicated doses of 0 properties that result in potent in vivo efficacy with ≤ 1 mg/kg/week IV administration. 0 50 100 150 200 CCGCCG 3.27 ±0.06 SU-DHL-10 DLBCL - GCB 7.3 + eFT226 (Q1W IV or IP). P-values (in parentheses) were calculated from the mean tumor volume relative Preclinical efficacy studies using eFT226 showed in vivo activity across hematological tumor Time (s) CAACAA + eFT226 2.43 ±0.01 VAL DLBCL - GCB 6.6 - to vehicle control using one-way ANOVA models suggesting that eFT226 may be active in tumor types where overexpression or CAACAA 3.78 ±0.05 Carnaval DLBCL - GCB 4.4 ++ U2973 DLBCL - GCB 4.2 + A. B. amplification of oncogenes such as c-MYC, CCND1/3, BCL2 or MCL-1 play a role in disease Tumor Growth Time Dependent Regulation of MYC Ramos Burkitt 4.6 ++ C. 1 mg/kg eFT226 1.5 pathogenesis. These results demonstrate the potential of eFT226 for the treatment of B- Jeko1 MCL 7.9 ++ 1.2

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r F163L 20 • eFT226 translationally regulates the protein expression of multiple 0 % 0.0001 0.001 0.01 0.1 1 0 oncogenic target genes (MYC, MCL1, BCL2) through the formation of • eIF4A RNA helicase is an essential component of the translation -25 DMSO 25nM 100 nM [eFT226], M initiation complex that regulates multiple key oncogenes involved in eFT226 a stable ternary complex [eIF4A-mRNA-eFT226] with sequence tumor cell proliferation, survival and metastasis specific recognition motifs in the 5’-UTR resulting in a block in tumor Figure 3. eIF4A1 binding site mutation rescues eFT226 anti-tumor activity. A) Downregulation of eFT226 Figure 6. eFT226 down regulates MCL1 and induces tumor cell death. A) Experimental design of cell proliferation and induction of cell death • B-cell receptor signaling activates pathways that enhance eIF4A activity sensitive genes is rescued with eIF4A1 F163L knock-in mutation. Cells were treated with eFT226 for 3 or competition experiment. B) MCL1 [ORF only] overexpression is insensitive to eFT226 treatment leading to the translation of NFkB target genes (i.e., c-MYC, BCL2) 24 hr and protein levels were analyzed by western blot. B) eIF4A1 F163L mutation rescues eFT226 anti- demonstrating a survival advantage in mixed populations. Ramos lymphoma cells were infected with • eFT226 is well-tolerated and shows efficacy against B-cell lymphoma proliferative activity linking the anti-tumor phenotype to eIF4A1 inhibition. Cells were treated with either Control-GFP or MCL1 [ORF only]-RPF lentiviral constructs. Cells mixed at a 1:1 ratio were treated xenograft models in vivo • eFFECTOR Therapeutics has designed eFT226, a potent, small molecule eFT226 for 72 hr and cell proliferation was assessed by CellTiter Glo. with various concentrations of eFT226 for 48 hr. The percent population of live cells was assessed by flow inhibitor of eIF4A cytometry. • Clinical trials with eFT226 in patients with hematological malignancies are planned

PAT, BE, NPY, SR, JC, MB, HH, AP, JM, JS, INJH, GSP, GGC, CJW, AN, JC, SS, AX, CT, CN, GKP, TM, PAS, JTE, SHR and KRW are employees and/or shareholders at eFFECTOR Therapeutics.