Tables S1-S3, Figures S1-S5

Flavaglines such as silvestrol (1) and rocaglamide (2) constitute an interesting class of natural products with promising anticancer activities. Their mode of action is based on inhibition of eukaryotic initiation factor 4A (eIF4A) dependent translation through formation of a stable ternary complex with eIF4A and mRNA, thus blocking ribosome scanning. Herein we describe initial SAR studies in a novel series of 1-aminomethyl substituted flavagline-inspired eIF4A inhibitors. We discovered that a variety of N-substitutions at the 1-aminomethyl group are tolerated, making this position pertinent for property and ADME profile tuning. The findings presented herein are relevant to future drug design efforts towards novel eIF4A inhibitors with drug-like properties.

Oncoprotein expression is controlled at the level of mRNA translation and is regulated by the eukaryotic translation initiation factor 4F (eIF4F) complex. eIF4A, a component of eIF4F, catalyzes the unwinding of secondary structure in the 5'-untranslated region (5'-UTR) of mRNA to facilitate ribosome scanning and translation initiation. Zotatifin (eFT226) is a selective eIF4A inhibitor that increases the affinity between eIF4A and specific polypurine sequence motifs and has been reported to inhibit translation of driver oncogenes in models of lymphoma. Here we report the identification of zotatifin binding motifs in the 5'-UTRs of HER2 and FGFR1/2 Receptor Tyrosine Kinases (RTKs). Dysregulation of HER2 or FGFR1/2 in human cancers leads to activation of the PI3K/AKT and RAS/ERK signaling pathways, thus enhancing eIF4A activity and promoting the translation of select oncogenes that are required for tumor cell growth and survival. In solid tumor models driven by alterations in HER2 or FGFR1/2, downregulation of oncoprotein expression by zotatifin induces sustained pathway-dependent anti-tumor activity resulting in potent inhibition of cell proliferation, induction of apoptosis, and significant in vivo tumor growth inhibition or regression. Sensitivity of RTK-driven tumor models to zotatifin correlated with high basal levels of mTOR activity and elevated translational capacity highlighting the unique circuitry generated by the RTK-driven signaling pathway. This dependency identifies the potential for rational combination strategies aimed at vertical inhibition of the PI3K/AKT/eIF4F pathway. Combination of zotatifin with PI3K or AKT inhibitors was beneficial across RTK-driven cancer models by blocking RTK-driven resistance mechanisms demonstrating the clinical potential of these combination strategies.

Abstract The PI3K/AKT/mTOR pathway is often activated in lymphoma through alterations in PI3K, PTEN, and B-cell receptor signaling, leading to dysregulation of eIF4A (through its regulators, eIF4B, eIF4G, and PDCD4) and the eIF4F complex. Activation of eIF4F has a direct role in tumorigenesis due to increased synthesis of oncogenes that are dependent on enhanced eIF4A RNA helicase activity for translation. eFT226, which inhibits translation of specific mRNAs by promoting eIF4A1 binding to 5′-untranslated regions (UTR) containing polypurine and/or G-quadruplex recognition motifs, shows potent antiproliferative activity and significant in vivo efficacy against a panel of diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma models with ≤1 mg/kg/week intravenous administration. Evaluation of predictive markers of sensitivity or resistance has shown that activation of eIF4A, mediated by mTOR signaling, correlated with eFT226 sensitivity in in vivo xenograft models. Mutation of PTEN is associated with reduced apoptosis in vitro and diminished efficacy in vivo in response to eFT226. In models evaluated with PTEN loss, AKT was stimulated without a corresponding increase in mTOR activation. AKT activation leads to the degradation of PDCD4, which can alter eIF4F complex formation. The association of eFT226 activity with PTEN/PI3K/mTOR pathway regulation of mRNA translation provides a means to identify patient subsets during clinical development.

Dysregulation of protein translation is a key driver for the pathogenesis of many cancers. Eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase, is a critical component of the eIF4F complex, which regulates cap-dependent protein synthesis. The flavagline class of natural products (i.e., rocaglamide A) has been shown to inhibit protein synthesis by stabilizing a translation-incompetent complex for select messenger RNAs (mRNAs) with eIF4A. Despite showing promising anticancer phenotypes, the development of flavagline derivatives as therapeutic agents has been hampered because of poor drug-like properties as well as synthetic complexity. A focused effort was undertaken utilizing a ligand-based design strategy to identify a chemotype with optimized physicochemical properties. Also, detailed mechanistic studies were undertaken to further elucidate mRNA sequence selectivity, key regulated target genes, and the associated antitumor phenotype. This work led to the design of eFT226 (Zotatifin), a compound with excellent physicochemical properties and significant antitumor activity that supports clinical development.

Abstract Mutations or amplifications affecting receptor tyrosine kinases (RTKs) activate the RAS/MAPK and PI3K/AKT signaling pathways thereby promoting cancer cell proliferation and survival. Oncoprotein expression is tightly controlled at the level of mRNA translation and is regulated by the eukaryotic translation initiation factor 4F (eIF4F) complex consisting of eIF4A, eIF4E, and eIF4G. eIF4A functions to catalyze the unwinding of secondary structure in the 5'-untranslated region (5'-UTR) of mRNA facilitating ribosome scanning and translation initiation. The activation of oncogenic signaling pathways, including RAS and PI3K, facilitate formation of eIF4F and enhance eIF4A activity promoting the translation of oncogenes with highly structured 5'-UTRs that are required for tumor cell proliferation, survival and metastasis. eFT226 is a selective eIF4A inhibitor that converts eIF4A into a sequence specific translational repressor by increasing the affinity between eIF4A and 5'-UTR polypurine motifs leading to selective downregulation of mRNA translation. The polypurine element is highly enriched in the 5'-UTR of eFT226 target genes, many of which are known oncogenic drivers, including FGFR1,2 and HER2, enabling eFT226 to selectively inhibit dysregulated oncogene expression. Formation of a ternary complex [eIF4A-eFT226-mRNA] blocks ribosome scanning along the 5'-UTR leading to dose dependent inhibition of RTK protein expression. The 5'-UTR sequence dependency of eFT226 translational inhibition was evaluated in cell-based reporter assays demonstrating 10-45-fold greater sensitivity for reporter constructs containing an RTK 5'-UTR compared to a control. In solid tumor cell lines driven by alterations in FGFR1, FGFR2 or HER2, downregulation of RTK expression by eFT226 resulted in decreased MAPK and AKT signaling, potent inhibition of cell proliferation and an induction of apoptosis suggesting that eFT226 could be effective in treating tumor types dependent on these oncogenic drivers. Solid tumor xenograft models harboring FGFR1,2 or HER2 amplifications treated with eFT226 resulted in significant in vivo tumor growth inhibition and regression at well tolerated doses in breast, non-small cell lung and colorectal cancer models. Treatment with eFT226 also decreased RTK protein levels supporting the potential to use these eFT226 target genes as pharmacodynamic markers of target engagement. Further evaluation of predictive markers of sensitivity or resistance showed that RTK tumor models with mTOR mediated activation of eIF4A are most sensitive to eFT226. The association of eFT226 activity in RTK tumor models with mTOR pathway activation provides a means to further enrich for sensitive patient subsets during clinical development. Clinical trials with eFT226 in patients with solid tumor malignancies have initiated. Citation Format: Peggy A. Thompson, Nathan P. Young, Adina Gerson-Gurwitz, Boreth Eam, Vikas Goel, Craig R. Stumpf, Joan Chen, Gregory S. Parker, Sarah Fish, Maria Barrera, Eric Sung, Jocelyn Staunton, Gary G. Chiang, Kevin R. Webster. Preclinical evaluation of eFT226, a potent and selective eIF4A inhibitor with anti-tumor activity in FGFR1,2 and HER2 driven cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3399.

Rocaglates, rocaglamides, and related flavagline natural products exert their remarkable anticancer activity through inhibition of eukaryotic initiation factor 4A (eIF4A) but generally display suboptimal drug-like properties. In our efforts to identify potent drug-like eIF4A inhibitors, we developed synthetic strategies for diastereoselectively functionalizing the C1 position of aza-rocaglamide scaffolds (cf. 14 and 18), which proceed via retention or inversion of configuration at C1 depending on the C2 substituent (cf. 15 and 21) and ultimately enabled the discovery of novel and potent eIF4A inhibitors such as 25.

Dysregulated messenger RNA (mRNA) translation drives the pathogenesis of multiple hematological malignancies. In lymphoma this includes the upregulation of key driver oncogenes and anti-apoptotic proteins (e.g., MYC, CCND1/3, BCL2 and MCL1) that contain a highly structured 5’-untranslated region (UTR) in their mRNA requiring enhanced eIF4A helicase activity for translation. eIF4A is a component of the eIF4F translation initiation complex and catalyzes the ATP-dependent unwinding of RNA duplexes and facilitates 43S ribosome scanning within the 5’-UTR. The activation of oncogenic signaling pathways, including RAS and PI3K, enhance eIF4A activity through phosphorylation of eIF4B, eIF4G and PDCD4 which facilitates formation of eIF4F and full activation of eIF4A. The PI3K/AKT/mTOR pathway is frequently activated in lymphoma, promoting the translation of oncogenes with complex 5’-UTRs that are required for tumor cell proliferation, survival and metastasis. eFT226 is a potent and sequence selective eIF4A1 inhibitor that promotes eIF4A1 binding to specific 5’-UTR polypurine and/or G-quadraplex recognition motifs leading to a selective block in ribosome mRNA scanning. The sequence dependency of eFT226 translational inhibition was evaluated in cell-based reporter assays demonstrating >100-fold greater sensitivity for 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 with increased drug residence time between eFT226, eIF4A1 and RNA oligonucleotides containing polypurine motifs. The ability of eFT226 to inhibit MYC or MCL1 expression was found to be dependent on the presence of their respective 5’-UTR supporting a translational regulation mechanism dependent on recognition elements within the 5’-UTR. eFT226 shows potent anti-proliferative activity (GI50 < 15 nM) against a panel of B-cell lymphoma cell lines. Treatment with eFT226 leads to coordinated inhibition of MYC, CCND1/3, BCL2 or MCL1 protein expression resulting in significant anti-tumor activity. eFT226 has good pharmacokinetic properties and exhibits significant in vivo activity across a panel of diffuse large B cell lymphoma (DLBCL), and Burkitt lymphoma tumor models with ≤1 mg/kg/week IV administration. Further evaluation of predictive markers of sensitivity or resistance has shown that tumors with mTOR mediated activation of eIF4A are most sensitive to eFT226. In addition, tumors with PTEN mutations do not exhibit activated eIF4A and are generally resistant to induction of apoptosis by eFT226, resulting in reduced in vivo efficacy. The association of eFT226 activity with PI3K/mTOR pathway activation and mutational status provides a means to identify patient subsets during clinical development. Clinical trials in patients with lymphoma and other malignancies are planned. Citation Format: Peggy A. Thompson, Boreth Eam, Nathan P. Young, Sarah Fish, Joan Chen, Maria Barrera, Haleigh Howard, Eric Sung, Ana Parra, Jocelyn Staunton, 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. eFT226, a potent and selective inhibitor of eIF4A, is efficacious in preclinical models of lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2698.

Abstract Background: Mutations or amplifications affecting receptor tyrosine kinases (RTKs) activate the RAS/MAPK and PI3K/AKT signaling pathways thereby promoting cancer cell proliferation and survival. Oncoprotein expression is tightly controlled at the level of mRNA translation and is regulated by the eukaryotic translation initiation factor 4F (eIF4F) complex consisting of eIF4A, eIF4E, and eIF4G. eIF4A functions to catalyze the unwinding of secondary structure in the 5’-untranslated region (5’-UTR) of mRNA facilitating ribosome scanning and translation initiation. eFT226 is a first in class inhibitor that converts eIF4A1 into a sequence specific translational repressor. eFT226 increases the affinity between eIF4A1 and polypurine recognition elements in the 5’-UTR leading to selective downregulation of mRNA translation. The polypurine element is highly enriched in the 5’-UTR of eFT226 target genes, many of which are known oncogenic drivers, including FGFR1/2 and HER2, enabling eFT226 to selectively inhibit dysregulated oncogene expression. Methods: 5’-UTR dependency was evaluated using cell-based luciferase reporter assays. Regulation of protein expression was analyzed by western blot analysis. Antitumor activity was assessed in vitro by proliferation and apoptosis assays. For in vivo experiments, athymic nude or NOD/SCID mice were implanted with subcutaneous xenograft models of FGFR1, FGFR2 or HER2 driven tumors and treated with eFT226 administered Q4D IV. Results: eFT226 inhibits the translation of FGFR1, FGFR2 and HER2 through formation of a sequence dependent ternary complex with eIF4A1 and polypurine elements within the 5’-UTR of mRNA [eFT226-eIF4A1-mRNA]. Formation of this ternary complex blocks ribosome scanning along the 5’-UTR leading to dose dependent inhibition of RTK protein expression. Cells transiently transfected with luciferase reporter constructs containing the 5’-UTR of each RTK resulted in 10-45-fold greater sensitivity to inhibition by eFT226 compared to a control 5’-UTR confirming the 5’-UTR dependency. In solid tumor cell lines driven by alterations in FGFR1, FGFR2 or HER2, downregulation of RTK expression by eFT226 resulted in decreased MAPK and AKT signaling, potent inhibition of cell proliferation and an induction of apoptosis suggesting that eFT226 could be effective in treating tumor types dependent on these oncogenic drivers. Solid tumor xenograft models harboring FGFR1/2 or HER2 amplifications treated with eFT226 resulted in significant in vivo tumor growth inhibition and regression at well tolerated doses in breast, non-small cell lung and colorectal cancer models. Treatment with eFT226 also decreased RTK protein levels supporting the potential to use these eFT226 target genes as pharmacodynamic markers of target engagement. Conclusions: eFT226 is efficacious against tumor models with alterations in FGFR1, FGFR2 and HER2 RTKs. The antitumor response observed in preclinical in vivo models driven by RTK amplifications demonstrates the potential for eFT226 in the treatment of solid tumors with FGFR1/2 or HER2 alterations. Furthermore, this data provides a means to select sensitive patient subsets during clinical development. Clinical trials in patients with solid tumor malignancies are planned. Citation Format: Peggy A Thompson, Nathan P Young, Craig R Stumpf, Boreth Eam, Vikas K Goel, Joan Chen, Sarah Fish, Gregory S Parker, Adina Gerson-Gurwitz, Maria Barrera, Eric Sung, Jocelyn Staunton, Gary G Chiang, Christopher J Wegerski, Samuel Sperry, Kevin R Webster, Siegfried H Reich. eFT226, a first in class inhibitor of eIF4A1, targets FGFR1/2 and HER2 driven cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B133. doi:10.1158/1535-7163.TARG-19-B133

Dysregulated translation of mRNA plays a major role in tumorigenesis. Mitogen-activated protein kinase interacting kinases (MNK)1/2 are key regulators of mRNA translation integrating signals from oncogenic and immune signaling pathways through phosphorylation of eIF4E and other mRNA binding proteins. Modulation of these key effector proteins regulates mRNA, which controls tumor/stromal cell signaling. Compound 23 (eFT508), an exquisitely selective, potent dual MNK1/2 inhibitor, was designed to assess the potential for control of oncogene signaling at the level of mRNA translation. The crystal structure-guided design leverages stereoelectronic interactions unique to MNK culminating in a novel pyridone aminal structure described for the first time in the kinase literature. Compound 23 has potent in vivo antitumor activity in models of diffuse large cell B-cell lymphoma and solid tumors, suggesting that controlling dysregulated translation has real therapeutic potential. Compound 23 is currently being evaluated in Phase 2 clinical trials in solid tumors and lymphoma. Compound 23 is the first highly selective dual MNK inhibitor targeting dysregulated translation being assessed clinically.

Abstract eFT226: A Selective and Highly Potent Inhibitor of Eukaryotic Initiation Factor 4A (eIF4A), a Novel Approach for the Treatment of Cancer Siegfried H Reich, Peggy A Thompson, Justin T Ernst, Boreth Eam, Nathan P Young, Sarah Fish, Joan Chen, Maria Barrera, Haleigh Howard, Ana Parra, Eric Sung, 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, and Kevin R Webster Effector Therapeutics, San Diego, CA Oncoprotein expression is tightly controlled at the level of RNA translation which is largely regulated by the eukaryotic translation initiation factor 4F (eIF4F). eIF4A1, a component of the eIF4F complex, catalyzes the ATP dependent unwinding of RNA duplexes and facilitates 43S ribosome complex scanning within the 5'-untranslated region (UTR). eIF4A1 is required for efficient translation of key oncogenes that contain complex secondary structures within the 5'-UTR. eFT226 is a novel, potent and selective eIF4A small molecule inhibitor with excellent physicochemical and pharmaceutical properties. The design of eFT226 involved ab initio ligand-based methods coupled with small molecule crystal structure analysis. eFT226 inhibits eIF4A1 through a reversible sequence-selective enhancement of eIF4A1 binding to mRNA with specific polypurine motifs within the 5'-UTR. The formation of a stable ternary complex [eIF4A1/eFT226/mRNA] with specific sequence recognition motifs leads to a block in ribosome scanning of select mRNAs. Treatment of lymphoma, AML, breast, colorectal, lung and hepatocellular tumor cell lines with eFT226 led to a dose dependent translational down regulation (IC50 of ~5-20 nM) of key oncogenes that drive tumor cell survival and proliferation (i.e., c-MYC, CCND1, BCL2 and MCL-1). Oncogene down regulation results in potent inhibition of cellular proliferation (GI50 of ~2-30 nM) across a panel of tumor cell lines. Enhanced anti-tumor sensitivity and a rapid induction of apoptosis was observed in hematological cell lines including lymphoma and AML. Inhibition of tumor cell proliferation and survival with eFT226 treatment results in significant antitumor activity in vivo in multiple human tumor models of DLBCL, Burkitt's lymphoma, acute myeloid leukemia (AML), and solid tumors following ≤ 1 mg/kg/week IV administration. eFT226 regulates the protein expression of multiple metabolic markers (i.e. c-MYC, HK2, TXNIP and GLUT1) that result in inhibition of tumor glucose uptake supporting the use of 18F-FDG-PET imaging as a measure of eFT226 target engagement in the clinic. These data demonstrate that eFT226 is a sequence-selective translational repressor of key oncogenic drivers that are essential for tumor cell proliferation and survival and support the clinical development of eFT226 in cancer patients. Citation Format: Siegfried H. Reich, Peggy A. Thompson, Justin T. Ernst, Boreth Eam, Nathan P. Young, Sarah Fish, Joan Chen, Maria Barrera, Haleigh Howard, Ana Parra, Eric Sung, 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, Kevin R. Webster. eFT226: A selective and highly potent inhibitor of eukaryotic initiation factor 4A (eIF4A), a novel approach for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr DDT02-05.

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.