Tumor Growth - effector.com€¦ · 14/12/2017 · Carnaval DLBCL - GCB 4.4 ++ U2973 DLBCL - GCB...
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DMSO 25nM 100 nM 0 20 40 60 80 100 % Live Population Cont_GFP MCL1_RFP eFT226 1 2 3 4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Log [eFT226], nM Relative CCND1 Levels (to GAPDH) eFT226 Exposure-Response Veh 1hr 3hr 24hr 72hr 0.0 0.5 1.0 1.5 Relative CCND1 Levels (to GAPDH) Time Dependent Regulation of CCND1 1 mg/kg eFT226 Veh 1hr 3hr 24hr 72hr 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Relative MYC Levels (to GAPDH) Time Dependent Regulation of MYC 1 mg/kg eFT226 0 5 10 15 0 500 1000 1500 2000 2500 3000 3500 Days on Tx Tumor Volume (mm 3 ) Tumor Growth Vehicle (D5W) Q1W IP eFT226 0.1 mg/kg Q1W IP eFT226 1 mg/kg Q1W IP 0 5 10 15 16 18 20 22 24 Body Weight Days of TX (Dosed on Day 1, 8, & 15) Body Weight (g) 0 5 10 15 0 500 1000 1500 2000 Days of TX (Dosed on Day 1, 8, & 15) Tumor Volume (mm 3 ) Vehicle eFT226 0.001mg/kg eFT226 0.2mg/kg eFT226 1mg/kg Tumor Growth 0.0001 0.001 0.01 0.1 1 -25 0 25 50 75 100 125 [eFT226], M % Growth relative to DMSO Parental F163L -4 -2 0 0.0 0.5 1.0 1.5 [eFT226] (M) RLU (Fold Change) CAA AGA GGC CCG 0 50 100 150 200 0 500 1000 1500 2000 Time (s) Response (RU) eIF4A only eIF4A + eFT226 F163 Preclinical Evaluation of eFT226, a Novel, Potent and Selective eIF4A Inhibitor with Anti-tumor 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 Translational control of oncoprotein expression has been implicated in the pathogenesis of multiple solid tumors and hematological malignancies. Protein synthesis is tightly regulated largely at the initiation stage through the eukaryotic translation initiation factor 4F (eIF4F) complex. eIF4A, an RNA helicase, is an essential component of the translation initiation complex and selectively regulates a subset of mRNAs based on the sequence and structure of the 5’-untranslated region (UTR). B-cell receptor signaling activates eIF4A resulting in the selective upregulation of oncogenes involved in cell proliferation, survival and metastasis. B-cell malignancies are often associated with dysregulation of oncogenes or anti-apoptotic proteins (e.g., c-MYC, CCND1/3, BCL2 and MCL-1) that contain structured 5’-UTRs and require enhanced eIF4A activity for translation. eFT226 is a potent and selective eIF4A inhibitor that promotes eIF4A binding to specific mRNA sequences and interferes with the assembly of the eIF4F initiation complex. eFT226 selectively inhibits translation of mRNAs containing longer 5’-UTRs, an increased frequency of uORFs (upstream open reading frame), and polypurine and/or G-quadraplex recognition motifs. The sequence dependency of eFT226 translational inhibition was evaluated in an in vitro translation assay demonstrating potent inhibition of reporter constructs containing a polypurine motif in the 5’-UTR (IC 50 ~2 nM). Direct binding studies also confirmed the formation of a stable ternary complex between eFT226, eIF4A and mRNA oligonucleotides containing polypurine motifs. This results in eFT226 selectively and translationally downregulating a subset of genes that are important for tumor growth and survival. eFT226 showed potent anti-proliferative activity (GI 50 < 15 nM) and an induction of apoptosis against a panel of B-cell lymphoma cell lines. Sensitivity to eFT226 was associated with dose-dependent decreases in the oncogenic drivers c-MYC, CCND1/3, BCL2 or MCL-1 in ABC (activated B-Cell) and GCB (germinal center B-cell) DLBCL (Diffuse Large B- cell Lymphoma) subtypes and Burkitt’s lymphoma. eFT226 has good pharmacokinetic properties that result in potent in vivo efficacy with ≤ 1 mg/kg/week IV administration. Preclinical efficacy studies using eFT226 showed in vivo activity across hematological tumor models suggesting that eFT226 may be active in tumor types where overexpression or amplification of oncogenes such as c-MYC, CCND1/3, BCL2 or MCL-1 play a role in disease pathogenesis. These results demonstrate the potential of eFT226 for the treatment of B- cell malignancies. Clinical trials in patients with hematological and other malignancies are planned. Introduction Results Conclusions Cell line Tumor type eFT226 GI 50 , nM Apoptosis TMD8 DLBCL - ABC 4.1 +++ SU-DHL-2 DLBCL - ABC 3 +++ HBL1 DLBCL - ABC 5.6 ++ Pfeiffer DLBCL - ABC 3.7 +++ SU-DHL-6 DLBCL - GCB 5.3 + SU-DHL-10 DLBCL - GCB 7.3 + VAL DLBCL - GCB 6.6 - Carnaval DLBCL - GCB 4.4 ++ U2973 DLBCL - GCB 4.2 + Ramos Burkitt 4.6 ++ Jeko1 MCL 7.9 ++ Mino MCL 11.2 + Rec-1 MCL 11.8 + AMO-1 MM 8.1 nt MM1.S MM 8.9 nt MM1.R MM 12.8 nt RPMI-8226 MM 5.9 nt SUPHD HL 7.7 nt HDLM2 HL 15.2 nt HS445 HL 3.8 nt KDMH2 HL 9.5 nt L428 HL 19.1 nt L1236 HL 17.4 nt eFT226 Selectively Binds to Free eIF4A Figure 2. eFT226 is a sequence specific translational regulator. A) eFT226 induces the formation of a stable ternary complex [eIF4A-RNA-eFT226]. Biacore sensograms of eIF4A1 binding to an AGAGAG RNA surface in the presence (green) or absence (black) or eFT226 shows that eFT226 increases the residence time for eIF4A with AGAGAG RNA. The dependence of ternary complex formation on the RNA sequence is summarized in the table on the right. Sensograms were fitted using a global fitting function to determine the equilibrium dissociation constants. B) Inhibition of in vitro translation by eFT226 is sequence dependent. Luciferase reporter gene constructs containing 5’-UTRs with 6-mer sequence motif repeats were transiently transfected into the MDA-MB-231 cell line and treated with increasing concentrations of eFT226 or hippuristanol for 4 hr in triplicate. Data were fitted to a four parameter dose response curve. Green: AGAGAG, Blue: GGCGGC, Red: CCGCCG and Black: CAACAA. IC 50 values are summarized in the table on the right. Figure 7. eFT226 inhibits the growth of B-cell lymphoma xenografts in vivo. A) TMD8 xenograft-bearing animals were treated with the indicated dose of eFT226 Q1W for 15 days. Left panel, tumor volume measurements; Right panel, body weight measurements taken throughout the tumor study. B) Table of tumor growth inhibition values at day 14-22 in lymphoma xenograft models for the indicated doses of eFT226 (Q1W IV or IP). P-values (in parentheses) were calculated from the mean tumor volume relative to vehicle control using one-way ANOVA A. A. A. B. Figure 8. Exposure-Response relationship of eFT226 in vivo. A) Pfeiffer xenograft-bearing animals were treated with the indicated dose of eFT226 for 14 days and tumor growth inhibition was measured. B-D) Pfeiffer xenograft-bearing animals were treated with 1mg/kg of eFT226 and tumor and plasma samples were harvested at time points (1-72 hr) post dose. B) CCND1 and C) MYC protein levels were measured by immunoblot and normalized relative to GAPDH. D) Relative CCND1 protein levels were plotted as a function of the corresponding eFT226 tumor concentration. B. A. C. • eIF4A RNA helicase is an essential component of the translation initiation complex that regulates multiple key oncogenes involved in tumor cell proliferation, survival and metastasis • B-cell receptor signaling activates pathways that enhance eIF4A activity leading to the translation of NFkB target genes (i.e., c-MYC, BCL2) • eFFECTOR Therapeutics has designed eFT226, a potent, small molecule inhibitor of eIF4A Xenograft Tumor type Genomic markers % Tumor growth inhibition (p-value) [1mg/kg eFT226] TMD8 DLBCL - ABC CD79, MYD88 97% (<0.0001) HBL1 DLBCL - ABC CD79, MYD88 87% (<0.0001) Pfeiffer DLBCL - GCB BCL2 Translocation, STAT3, EZH2 70% (0.0001) SU-DHL-6 DLBCL - GCB BCL2 Translocation, EGFR, EZH2, TP53 83% (0.0002) SU-DHL-10 DLBCL - GCB MYC and BCL2 Translocations, EZH2, PTEN, TP53 37% (0.06) Ramos Burkitt’s MYC Translocation, IL2, PDGFRB, TP53 75% (<0.0001) #1530 D. 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. • eFT226 is a potent and selective inhibitor of eIF4A1 • eFT226 translationally regulates the protein expression of multiple oncogenic target genes (MYC, MCL1, BCL2) through the formation of a stable ternary complex [eIF4A-mRNA-eFT226] with sequence specific recognition motifs in the 5’-UTR resulting in a block in tumor cell proliferation and induction of cell death • eFT226 is well-tolerated and shows efficacy against B-cell lymphoma xenograft models in vivo • Clinical trials with eFT226 in patients with hematological malignancies are planned 5’-UTR Sequence eFT226 IC 50 (nM) Hippuristanol IC 50 , nM AGAGAG 1.5 ±0.4 426 GGCGGC 13.8 ±2.0 204 CCGCCG 92.2 ±17.8 369 CAACAA 217.5 ±55 210 RNA Sequence K D (M) AGAGAG + eFT226 0.021 ±0.001 AGAGAG 8.0 ±0.9 GGCGGC + eFT226 3.19 ±0.03 GGCGGC 8.0 ±0.3 CCGCCG + eFT226 9.6 ±0.4 CCGCCG 3.27 ±0.06 CAACAA + eFT226 2.43 ±0.01 CAACAA 3.78 ±0.05 Figure 1. eFT226 forms a ternary complex with free eIF4A1. A) The eIF4A F163L binding site mutation was introduced using CRISPR gene editing. B) Pulldown experiments using biotinylated eFT226 shows selective binding to free eIF4A1 wt not the eFI4F complex. eIF4A1, 4E, 4B and 4B protein levels were analyzed by western blot. 200M unlabeled eFT226 was used to compete with the biotinylated-eFT226 binding. The eIF4A binding site mutation blocks interaction of eFT226 with eIF4A. eFT226 is a Sequence Specific Translational Regulator Figure 6. eFT226 down regulates MCL1 and induces tumor cell death. A) Experimental design of competition experiment. B) MCL1 [ORF only] overexpression is insensitive to eFT226 treatment demonstrating a survival advantage in mixed populations. Ramos lymphoma cells were infected with either Control-GFP or MCL1 [ORF only]-RPF lentiviral constructs. Cells mixed at a 1:1 ratio were treated with various concentrations of eFT226 for 48 hr. The percent population of live cells was assessed by flow cytometry. Figure 4. Protein expression of key oncogenes is regulated by eFT226 through the 5’-UTR. Doxycycline-inducible constructs A) MYC [ORF only] or MYC [5’-UTR + ORF] and B) MCL1 [ORF only] or MCL1 [5’-UTR + ORF] were over expressed in the HEK293T cell line. Cells were treated with eFT226 for 3 hr. MYC, MCL1, CCND1 and GAPDH protein levels were analyzed by western blot analysis. Sequence analysis of the 5’-UTR identified polypurine motifs similar to the high affinity binding sites identified by direct binding providing potential interaction sites for eFT226-induced ternary complex formation and translational regulation. A. B. Figure 5. eFT226 translationally regulates key oncogenic drivers resulting in inhibition of growth and survival. A) Tabulated summary of eFT226 GI 50 values and induction of apoptosis for the indicated cell lines. Growth inhibition was analyzed by CTG assay after treating cells for 72 hr with eFT226; apoptosis was assessed by Annexin V and PI staining of tumor cells and analyzed by flow cytometry after 24 hr treatment with eFT226 (apoptotic signature: - [weak] to +++ [strong]). B) Treatment of TMD8 and Pfeiffer DLBCL tumor cells with eFT226 for 3 and 24 hr results in translational regulation of oncogenic protein levels as assessed by western blot analysis. Depletion of protein levels after eFT226 treatment was unaffected by pretreatment with 10 nM bortezomib (BTZ) for 2 hr. eFT226 treatment does not inhibit mRNA levels further supporting the mechanism of translational regulation (data not shown). C) GSEA plot and heatmap depicting the down-regulation of a B cell line derived MYC signature in TMD8 cells following 50 nM eFT226 treatment for 6 hr using RNA-Seq analysis. NES, normalized enrichment score; na, not tested; Burkitt, Burkitt lymphoma; DLBCL, diffuse large B-cell lymphoma; HL, Hodgkin lymphoma; MCL, mantle cell lymphoma; MM, multiple myeloma. Frequent mutations in DLBCL eIF4E eIF4G eIF4A m 7 G ~20% Card11 ~10% ~35% IRAK1/4 CD19 BCR A20 ~20% NFκB IKK PI3Kδ BTK SYK MyD88 CD79 IL1/TLR AKT RTK PI3K mTOR PTEN S6K ~10% P eIF4B P PDCD4 Activator Inhibitor A. B. A. B. C. B. B. Inhibition of Key Oncogenes by eFT226 is Mediated Through the 5’-UTR eFT226 Inhibits Growth and Induces Apoptosis in Tumor Cell Lines In vivo Tumor Growth Inhibition by eFT226 226 + mRNA eIF4A 226 Stable ternary complex with free eIF4A Translation of NFkB target genes: • c-MYC • BCL2 eIF4A1 Binding Site Mutation Rescues eFT226 Anti-Tumor Activity Figure 3. eIF4A1 binding site mutation rescues eFT226 anti-tumor activity. A) Downregulation of eFT226 sensitive genes is rescued with eIF4A1 F163L knock-in mutation. Cells were treated with eFT226 for 3 or 24 hr and protein levels were analyzed by western blot. B) eIF4A1 F163L mutation rescues eFT226 anti- proliferative activity linking the anti-tumor phenotype to eIF4A1 inhibition. Cells were treated with eFT226 for 72 hr and cell proliferation was assessed by CellTiter Glo. Binding to AGAGAG RNA surface PDB ID: 2G9N A. B.
Transcript of Tumor Growth - effector.com€¦ · 14/12/2017 · Carnaval DLBCL - GCB 4.4 ++ U2973 DLBCL - GCB...
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Preclinical Evaluation of eFT226, a Novel, Potent and Selective eIF4A Inhibitor with Anti-tumor 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 Translational control of oncoprotein expression has been implicated in the pathogenesis of multiple solid tumors and hematological malignancies. Protein synthesis is tightly regulated largely at the initiation stage through the eukaryotic translation initiation factor 4F (eIF4F) complex. eIF4A, an RNA helicase, is an essential component of the translation initiation complex and selectively regulates a subset of mRNAs based on the sequence and structure of the 5’-untranslated region (UTR). B-cell receptor signaling activates eIF4A resulting in the selective upregulation of oncogenes involved in cell proliferation, survival and metastasis. B-cell malignancies are often associated with dysregulation of oncogenes or anti-apoptotic proteins (e.g., c-MYC, CCND1/3, BCL2 and MCL-1) that contain structured 5’-UTRs and require enhanced eIF4A activity for translation.
eFT226 is a potent and selective eIF4A inhibitor that promotes eIF4A binding to specific mRNA sequences and interferes with the assembly of the eIF4F initiation complex. eFT226 selectively inhibits translation of mRNAs containing longer 5’-UTRs, an increased frequency of uORFs (upstream open reading frame), and polypurine and/or G-quadraplex recognition motifs. The sequence dependency of eFT226 translational inhibition was evaluated in an in vitro translation assay demonstrating potent inhibition of reporter constructs containing a polypurine motif in the 5’-UTR (IC50 ~2 nM). Direct binding studies also confirmed the formation of a stable ternary complex between eFT226, eIF4A and mRNA oligonucleotides containing polypurine motifs. This results in eFT226 selectively and translationally downregulating a subset of genes that are important for tumor growth and survival. eFT226 showed potent anti-proliferative activity (GI50 < 15 nM) and an induction of apoptosis against a panel of B-cell lymphoma cell lines. Sensitivity to eFT226 was associated with dose-dependent decreases in the oncogenic drivers c-MYC, CCND1/3, BCL2 or MCL-1 in ABC (activated B-Cell) and GCB (germinal center B-cell) DLBCL (Diffuse Large B-cell Lymphoma) subtypes and Burkitt’s lymphoma. eFT226 has good pharmacokinetic properties that result in potent in vivo efficacy with ≤ 1 mg/kg/week IV administration. Preclinical efficacy studies using eFT226 showed in vivo activity across hematological tumor models suggesting that eFT226 may be active in tumor types where overexpression or amplification of oncogenes such as c-MYC, CCND1/3, BCL2 or MCL-1 play a role in disease pathogenesis. These results demonstrate the potential of eFT226 for the treatment of B-cell malignancies. Clinical trials in patients with hematological and other malignancies are planned.
Introduction
Results
Conclusions
Cell line Tumor type eFT226
GI50, nM Apoptosis
TMD8 DLBCL - ABC 4.1 +++
SU-DHL-2 DLBCL - ABC 3 +++
HBL1 DLBCL - ABC 5.6 ++
Pfeiffer DLBCL - ABC 3.7 +++
SU-DHL-6 DLBCL - GCB 5.3 +
SU-DHL-10 DLBCL - GCB 7.3 +
VAL DLBCL - GCB 6.6 -
Carnaval DLBCL - GCB 4.4 ++
U2973 DLBCL - GCB 4.2 +
Ramos Burkitt 4.6 ++
Jeko1 MCL 7.9 ++
Mino MCL 11.2 +
Rec-1 MCL 11.8 +
AMO-1 MM 8.1 nt
MM1.S MM 8.9 nt
MM1.R MM 12.8 nt
RPMI-8226 MM 5.9 nt
SUPHD HL 7.7 nt
HDLM2 HL 15.2 nt
HS445 HL 3.8 nt
KDMH2 HL 9.5 nt
L428 HL 19.1 nt
L1236 HL 17.4 nt
eFT226 Selectively Binds to Free eIF4A
Figure 2. eFT226 is a sequence specific translational regulator. A) eFT226 induces the formation of a stable ternary complex [eIF4A-RNA-eFT226]. Biacore sensograms of eIF4A1 binding to an AGAGAG RNA surface in the presence (green) or absence (black) or eFT226 shows that eFT226 increases the residence time for eIF4A with AGAGAG RNA. The dependence of ternary complex formation on the RNA sequence is summarized in the table on the right. Sensograms were fitted using a global fitting function to determine the equilibrium dissociation constants. B) Inhibition of in vitro translation by eFT226 is sequence dependent. Luciferase reporter gene constructs containing 5’-UTRs with 6-mer sequence motif repeats were transiently transfected into the MDA-MB-231 cell line and treated with increasing concentrations of eFT226 or hippuristanol for 4 hr in triplicate. Data were fitted to a four parameter dose response curve. Green: AGAGAG, Blue: GGCGGC, Red: CCGCCG and Black: CAACAA. IC50 values are summarized in the table on the right.
Figure 7. eFT226 inhibits the growth of B-cell lymphoma xenografts in vivo. A) TMD8 xenograft-bearing animals were treated with the indicated dose of eFT226 Q1W for 15 days. Left panel, tumor volume measurements; Right panel, body weight measurements taken throughout the tumor study. B) Table of tumor growth inhibition values at day 14-22 in lymphoma xenograft models for the indicated doses of eFT226 (Q1W IV or IP). P-values (in parentheses) were calculated from the mean tumor volume relative to vehicle control using one-way ANOVA
A.
A.
A.
B.
Figure 8. Exposure-Response relationship of eFT226 in vivo. A) Pfeiffer xenograft-bearing animals were treated with the indicated dose of eFT226 for 14 days and tumor growth inhibition was measured. B-D) Pfeiffer xenograft-bearing animals were treated with 1mg/kg of eFT226 and tumor and plasma samples were harvested at time points (1-72 hr) post dose. B) CCND1 and C) MYC protein levels were measured by immunoblot and normalized relative to GAPDH. D) Relative CCND1 protein levels were plotted as a function of the corresponding eFT226 tumor concentration.
B.
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C.
• eIF4A RNA helicase is an essential component of the translation initiation complex that regulates multiple key oncogenes involved in tumor cell proliferation, survival and metastasis
• B-cell receptor signaling activates pathways that enhance eIF4A activity leading to the translation of NFkB target genes (i.e., c-MYC, BCL2)
• eFFECTOR Therapeutics has designed eFT226, a potent, small molecule inhibitor of eIF4A
Xenograft Tumor type Genomic markers
% Tumor growth inhibition (p-value)
[1mg/kg eFT226]
TMD8 DLBCL - ABC CD79, MYD88 97% (<0.0001)
HBL1 DLBCL - ABC CD79, MYD88 87% (<0.0001)
Pfeiffer DLBCL - GCB BCL2 Translocation, STAT3, EZH2 70% (0.0001)
SU-DHL-6 DLBCL - GCB BCL2 Translocation, EGFR, EZH2, TP53 83% (0.0002)
SU-DHL-10 DLBCL - GCB MYC and BCL2 Translocations, EZH2, PTEN, TP53
37% (0.06)
Ramos Burkitt’s MYC Translocation, IL2, PDGFRB, TP53 75% (<0.0001)
#1530
D.
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.
• eFT226 is a potent and selective inhibitor of eIF4A1
• eFT226 translationally regulates the protein expression of multiple oncogenic target genes (MYC, MCL1, BCL2) through the formation of a stable ternary complex [eIF4A-mRNA-eFT226] with sequence specific recognition motifs in the 5’-UTR resulting in a block in tumor cell proliferation and induction of cell death
• eFT226 is well-tolerated and shows efficacy against B-cell lymphoma xenograft models in vivo
• Clinical trials with eFT226 in patients with hematological malignancies are planned
5’-UTR Sequence
eFT226 IC50 (nM)
Hippuristanol IC50, nM
AGAGAG 1.5 ±0.4 426
GGCGGC 13.8 ±2.0 204
CCGCCG 92.2 ±17.8 369
CAACAA 217.5 ±55 210
RNA Sequence KD (M)
AGAGAG + eFT226 0.021 ±0.001
AGAGAG 8.0 ±0.9
GGCGGC + eFT226 3.19 ±0.03
GGCGGC 8.0 ±0.3
CCGCCG + eFT226 9.6 ±0.4
CCGCCG 3.27 ±0.06
CAACAA + eFT226 2.43 ±0.01
CAACAA 3.78 ±0.05
Figure 1. eFT226 forms a ternary complex with free eIF4A1. A) The eIF4A F163L binding site mutation was introduced using CRISPR gene editing. B) Pulldown experiments using biotinylated eFT226 shows selective binding to free eIF4A1wt not the eFI4F complex. eIF4A1, 4E, 4B and 4B protein levels were analyzed by western blot. 200M unlabeled eFT226 was used to compete with the biotinylated-eFT226 binding. The eIF4A binding site mutation blocks interaction of eFT226 with eIF4A.
eFT226 is a Sequence Specific Translational Regulator
Figure 6. eFT226 down regulates MCL1 and induces tumor cell death. A) Experimental design of competition experiment. B) MCL1 [ORF only] overexpression is insensitive to eFT226 treatment demonstrating a survival advantage in mixed populations. Ramos lymphoma cells were infected with either Control-GFP or MCL1 [ORF only]-RPF lentiviral constructs. Cells mixed at a 1:1 ratio were treated with various concentrations of eFT226 for 48 hr. The percent population of live cells was assessed by flow cytometry.
Figure 4. Protein expression of key oncogenes is regulated by eFT226 through the 5’-UTR. Doxycycline-inducible constructs A) MYC [ORF only] or MYC [5’-UTR + ORF] and B) MCL1 [ORF only] or MCL1 [5’-UTR + ORF] were over expressed in the HEK293T cell line. Cells were treated with eFT226 for 3 hr. MYC, MCL1, CCND1 and GAPDH protein levels were analyzed by western blot analysis. Sequence analysis of the 5’-UTR identified polypurine motifs similar to the high affinity binding sites identified by direct binding providing potential interaction sites for eFT226-induced ternary complex formation and translational regulation.
A.
B.
Figure 5. eFT226 translationally regulates key oncogenic drivers resulting in inhibition of growth and survival. A) Tabulated summary of eFT226 GI50 values and induction of apoptosis for the indicated cell lines. Growth inhibition was analyzed by CTG assay after treating cells for 72 hr with eFT226; apoptosis was assessed by Annexin V and PI staining of tumor cells and analyzed by flow cytometry after 24 hr treatment with eFT226 (apoptotic signature: - [weak] to +++ [strong]). B) Treatment of TMD8 and Pfeiffer DLBCL tumor cells with eFT226 for 3 and 24 hr results in translational regulation of oncogenic protein levels as assessed by western blot analysis. Depletion of protein levels after eFT226 treatment was unaffected by pretreatment with 10 nM bortezomib (BTZ) for 2 hr. eFT226 treatment does not inhibit mRNA levels further supporting the mechanism of translational regulation (data not shown). C) GSEA plot and heatmap depicting the down-regulation of a B cell line derived MYC signature in TMD8 cells following 50 nM eFT226 treatment for 6 hr using RNA-Seq analysis. NES, normalized enrichment score; na, not tested; Burkitt, Burkitt lymphoma; DLBCL, diffuse large B-cell lymphoma; HL, Hodgkin lymphoma; MCL, mantle cell lymphoma; MM, multiple myeloma.
Frequent mutations
in DLBCL eIF4E
eIF4G eIF4A
m7G
~20%
Card11
~10%
~35%
IRAK1/4
CD19 BCR
A20
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PI3Kδ
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79
IL1/TLR
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PI3K
mTOR
PTEN
S6K
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P
PDCD4
Activator Inhibitor
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A. B.
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B.
Inhibition of Key Oncogenes by eFT226 is Mediated Through the 5’-UTR
eFT226 Inhibits Growth and Induces Apoptosis in Tumor Cell Lines
In vivo Tumor Growth Inhibition by eFT226
226 +
mRNA
eIF4A
226 Stable ternary complex
with free eIF4A
Translation of NFkB target genes:
• c-MYC
• BCL2
eIF4A1 Binding Site Mutation Rescues eFT226 Anti-Tumor Activity
Figure 3. eIF4A1 binding site mutation rescues eFT226 anti-tumor activity. A) Downregulation of eFT226 sensitive genes is rescued with eIF4A1 F163L knock-in mutation. Cells were treated with eFT226 for 3 or 24 hr and protein levels were analyzed by western blot. B) eIF4A1 F163L mutation rescues eFT226 anti-proliferative activity linking the anti-tumor phenotype to eIF4A1 inhibition. Cells were treated with eFT226 for 72 hr and cell proliferation was assessed by CellTiter Glo.
Binding to AGAGAG RNA surface
PDB ID: 2G9N
A. B.
