Abstract Background Zotatifin (eFT226) is a first-in-class, potent and sequence selective inhibitor of RNA helicase eIF4A that promotes a stable mRNA:eIF4A:drug ternary complex at specific polypurine motifs present within the 5’-UTR of select transcripts, thereby blocking production of the encoded proteins. In preclinical models zotatifin treatment simultaneously down-regulated translation of numerous oncogenes including CDK4, ERα, ERBB2, KRAS, and CCND1. Previous clinical research has demonstrated the activity of zotatifin in combination with other agents in ER+ metastatic breast cancer (MBC). Methods Part 1 is a 3+3 dose escalation portion of the protocol followed by Part 2 as a Simon’s two-stage design in patients with ER+ MBC enrolled at the recommended phase 2 dose (RP2D) of zotatifin (0.07 mg/kg IV two weeks on/one week off) in combination with fulvestrant (ful) and abemaciclib (abema) (Z+F+A). Part 1a and Part 1b are 3+3 dose escalation portions evaluating zotatifin in combination with ful (Z+F) at QW IV and Q2W IV dosing schedules, respectively. Key eligibility criteria included at least one line of therapy for advanced/metastatic disease, progression on hormone therapy, and in addition, the Z+F cohorts required prior CDK 4/6 inhibitor (CDKi). Primary endpoint of Part 1a/1b was determination of RP2D and primary endpoint of part 2 was objective response rate (ORR) per RECIST v1.1. Additional endpoints included safety, progression-free survival (PFS) and other efficacy analyses, and characterization of pharmacodynamic (PD) markers and pharmacokinetics. Results As of a data cut-off of July 6, 2023, 20 pts were enrolled in the Z+F+A cohort and had a median of four prior metastatic lines of therapy. In 19 evaluable pts in Z+F+A cohort, there were four confirmed partial responses (PR), one unconfirmed PR, and 10 pts with best overall response (OR) of stable disease (SD). All pts with PR/unconfirmed PR received prior CDKI, ful, and chemotherapy for MBC. Confirmed PRs were observed in patients with and without mutations in ESR1 and PIK3CA. mPFS, while not yet mature, exceeds 20 weeks. In Z+F+A the most common adverse events (AEs) were diarrhea (80%, all grades (Gr)) and nausea (75%, all Gr 1/2) and the most common Gr 3/4 AE was diarrhea (15%). In Z+F cohorts of 0.07 mg/kg QW (n=3), 0.1 mg/kg Q2W (n=3), and 0.14 Q2W (n= 2) there were no dose-limiting toxicities or Gr 5 adverse events (AEs). There was one confirmed PR and one SD in 0.1 and 0.14 mg/kg Q2W cohorts, respectively, and no PR or SD in the 0.07 mg/kg QW cohort. In Z+F+A, 9 pts had paired pre- and on-treatment ctDNA analyzed by Guardant assay. Of the remaining 11 pts, 4 came off study prior to scheduled collection of the on-treatment sample and 5 had unavailable samples. Of the 9 patients with paired samples, 8 had ctDNA decreases of >50%, all of whom had SD or PR. In the Z+F Q2W cohort, 2 of 3 patients had ctDNA decreases >50%, one of whom had a PR. Reduction below the limit of detection was seen for mutant alleles in ESR1, PIK3CA and other genes with potential to confer resistance to endocrine therapy. Conclusion Z+F+A showed encouraging efficacy in heavily pretreated ER+ MBC patients, with results that compare favorably to other treatment options available after progression on CDKi plus endocrine treatment. Efficacy was seen independent of ESR1 or PIK3CA mutations, suggesting the potential to treat a broad, unrestricted patient population. Initial results with Z+F Q2W dosing are encouraging and support continued dose escalation of this regimen. Citation Format: Ezra Y. Rosen, Manish Sharma, David Berz, Jennifer Caswell-Jin, Georgina Fulgar, Mark Densel, Gary Chiang, Samuel Sperry, Douglas Warner, Funda Meric-Bernstam. Phase 1/2 Dose Expansion Study Evaluating First-In-Class eIF4A Inhibitor Zotatifin in Patients with ER+ Metastatic Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-05-03.

1080 Background: Zotatifin (eFT226) is a first-in-class, potent and sequence selective inhibitor of RNA helicase eIF4A that promotes a stable mRNA:eIF4A:drug ternary complex at specific polypurine motifs within the 5’-UTR, preventing translation of select transcripts. In preclinical models zotatifin treatment simultaneously down-regulated translation of numerous oncogenes including CDK4, ERα, ERBB2, KRAS, and CCND1. Methods: Following a 3+3 dose escalation portion of the protocol (Part 1), Part 2 is a Simon’s two-stage design of cohorts of up to 18 patients (pts) with ER+ metastatic breast cancer enrolled at the recommended phase 2 dose of zotatifin (0.07 mg/kg IV two weeks on/one week off) in combination with fulvestrant (ful) (ECBF: Expansion, Combination, Breast, Ful) or in combination with ful and abemaciclib (abema) (ECBF+A: Expansion, Combination, Breast, Ful + Abema). Key eligibility criteria included at least one line of therapy for advanced/metastatic disease, progression on hormone therapy, and in addition, the ECBF cohort required prior CDK 4/6 inhibitor (CDKI). Primary endpoint was objective response rate (ORR) per RECIST v1.1. Additional endpoints included safety, other efficacy analyses, and for Parts 1 and 2, characterization of pharmacodynamic (PD) markers and pharmacokinetics. Results: As of a data cut-off of Jan 31, 2023, 18 pts were enrolled in each cohort. Median prior metastatic lines of therapy were 3 and 7 in ECBF+A and ECBF, respectively. In 16 evaluable pts in ECBF+A cohort, there were two confirmed partial responses (PR), two additional PRs awaiting confirmatory scans, and 8 pts with best overall response (OR) of stable disease (SD). All pts with PR/unconfirmed PR received prior CDKI and ful, including one pt with disease progression on combination abema and ful as the last therapy prior to enrollment. In 18 evaluable pts in ECBF, there was one confirmed PR and six SD as best OR. There were no dose-limiting toxicities or grade (Gr) 5 adverse events (AEs) in either cohort. In ECBF+A the most common AEs (all Gr 1/2) were diarrhea (44%) and nausea (31%) and 21% of pts had Gr 3/4 AEs. The most common AEs in ECBF (all Gr 1/2) were nausea (39%) and constipation (27%), and 28% of pts had Gr 3/4 AEs. Blood-based PD markers and ctDNA in Part 1 showed dose-dependent evidence of target engagement and anti-tumor activity. Conclusions: In dose expansion cohorts of heavily pre-treated metastatic breast cancer pts, eIF4A inhibitor zotatifin showed evidence of anti-tumor activity in combination with fulvestrant and abemaciclib and had a favorable safety profile consisting of primarily grade 1/2 adverse events. Clinical trial information: NCT04092673 .

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.

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 Aberrant protein translation plays a role in the pathogenesis of multiple solid tumors and hematologic malignancies. The translation initiation factor eIF4E is essential for the translation of m7G-capped mRNA and is a key point of convergence for several signaling pathways, such as PI3K/mTOR and MAPK, which are intimately involved in tumor cell growth and survival. As such, eIF4E has generated intense interest as a target for anticancer drug discovery. We have designed a series of potent, selective, and orally available m7G cap-competitive inhibitors of eIF4E (eFT-4Ei) with favorable drug-like properties. These inhibitors bind free eIF4E, eIF4E-4EBP and eIF4E-eIF4F complexes within tumor cells. Ribosomal profiling of eIF4E inhibitor-treated tumor cells has identified a subset of translationally regulated target genes that overlap with mTORC1/2 regulated genes, but also include a larger set of unique translationally regulated target mRNAs that are enriched for 5'-TOP, PRTE and CERT sequence elements in their 5'-untranslated regions. eIF4E inhibition results in potent antiproliferative activity and induction of apoptosis in a subset of tumor cell lines. Consistent with this observation, our eIF4E inhibitors show some similarities, yet several important differences from existing mTORC1 or mTORC1/2 dual inhibitors in both cellular and physiologic assays. Finally, significant antitumor efficacy was observed with eIF4E inhibition in both solid tumor and hematologic xenografts in vivo. Taken together, these results highlight the potential of targeting eIF4E as a novel and differentiated therapeutic strategy to treat cancer. Citation Format: Gregory S. Parker, Ivy N.J. Hung, Jocelyn Staunton, Maria Barrera, Eric Sung, Ana Parra, Craig R. Stumpf, Joan Chen, Peggy A. Thompson, Andreas Nevarez, Christopher J. Wegerski, Jeff Clarine, Samuel Sperry, Alan Xiang, Christian Nilewski, Garrick K. Packard, Kaveri Urklalan, Takasuke Mukaiyama, Theo Michels, Justin T. Ernst, Paul A. Sprengeler, Siegfried H. Reich, Gary G. Chiang, Kevin R. Webster. Targeting PI3K/mTOR signaling with potent, selective and orally-available small-molecule inhibitors of eIF4E [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B33.

Aberrant protein translation plays a role in the pathogenesis of multiple solid tumors and hematologic malignancies. The translation initiation factor eIF4E is essential for the translation of m7G-capped mRNA and is a key point of convergence for several signaling pathways, such as PI3K/mTOR and MAPK, which are intimately involved in tumor cell growth and survival. As such, eIF4E has generated intense interest as a target for anticancer drug discovery. We have designed a series of potent, selective, and orally available m7G cap-competitive inhibitors of eIF4E (eFT-4Ei) with favorable drug-like properties. These inhibitors bind free eIF4E, eIF4E-4EBP and eIF4E-eIF4F complexes within tumor cells. Ribosomal profiling of eIF4E inhibitor-treated tumor cells has identified a subset of translationally regulated target genes that overlap with mTORC1/2 regulated genes, but also include a larger set of unique translationally regulated target mRNAs that are enriched for 59-TOP, PRTE and CERT sequence elements in their 59-untranslated regions. eIF4E inhibition results in potent antiproliferative activity and induction of apoptosis in a subset of tumor cell lines. Consistent with this observation, our eIF4E inhibitors show some similarities, yet several important differences from existing mTORC1 or mTORC1/2 dual inhibitors in both cellular and physiologic assays. Finally, significant antitumor efficacy was observed with eIF4E inhibition in both solid tumor and hematologic xenografts in vivo. Taken together, these results highlight the potential of targeting eIF4E as a novel and differentiated therapeutic strategy to treat cancer. Citation Format: Gregory S. Parker, Ivy N.J. Hung, Jocelyn Staunton, Maria Barrera, Eric Sung, Ana Parra, Craig R. Stumpf, Joan Chen, Peggy A. Thompson, Andreas Nevarez, Christopher J. Wegerski, Jeff Clarine, Samuel Sperry, Alan Xiang, Christian Nilewski, Garrick K. Packard, Kaveri Urklalan, Takasuke Mukaiyama, Theo Michels, Justin T. Ernst, Paul A. Sprengeler, Siegfried H. Reich, Gary G. Chiang, Kevin R. Webster. Targeting PI3K/mTOR signaling with potent, selective and orally-available small-molecule inhibitors of eIF4E [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B33.

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

The PI3K/mTOR pathway is commonly dysregulated in many hormone receptor-dependent tumors and plays a key role in promoting tumor growth and mediating drug resistance. In particular, PI3K and mTORC1/2 inhibitors have been intensively studied in the treatment of hormone receptor-dependent cancers and have shown benefit in some clinical settings. However, issues such as dose-limiting toxicities and emergent resistance limit the broader utility of these inhibitors. The translation initiation factor eIF4E is essential for the translation of m7G-capped mRNA and is a key point of convergence for both the PI3K/mTOR and MAPK signaling pathways. We have designed a series of potent, selective and orally-available m7G cap-competitive inhibitors of eIF4E (eFT-4Ei) with favorable drug-like properties. These inhibitors bind to eIF4E either as its free form or with eIF4E-4EBP and eIF4F complexes within tumor cells and downregulate hormone receptor-dependent signaling. Ribosomal profiling of eIF4E inhibitor-treated tumor cells identified a subset of translationally regulated target genes that overlap with mTORC1/2 regulated genes, but also a unique set of translationally regulated target mRNAs. Consistent with this observation, our eIF4E inhibitors show some similarities yet several important differences from existing mTORC1 or mTORC1/2 dual inhibitors in both cellular and physiological assays. Finally, significant anti-tumor efficacy was observed with eIF4E inhibition in vitro and in vivo. Taken together, these results highlight the potential for targeting eIF4E as a novel therapeutic strategy to treat hormone-receptor dependent cancers. Citation Format: Gary G. Chiang, Gregory S. Parker, Ivy N. Hung, Vikas K. Goel, Jocelyn Staunton, Maria Barrera, Eric Sung, Ana Parra, Craig R. Stumpf, Joan Chen, Peggy A. Thompson, Andreas Nevarez, Christopher J. Wegerski, Cody Parker, Jeff Clarine, Samuel Sperry, Alan Xiang, Christian Nilewski, Garrick K. Packard, Kaveri Urkalan, Takasuke Mukaiyama, Theo Michels, Justin T. Ernst, Paul A. Sprengeler, Siegfried H. Reich, Kevin R. Webster. Targeting hormone receptor-dependent cancers with potent, selective and orally-available small molecule inhibitors of eIF4E [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 1302.

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.

Abstract Purpose: This study was designed to evaluate the potential of eFT508 to selectively regulate key immune signaling pathways and enhance anti-tumor immunity as a monotherapy or in combination with checkpoint blockade in immunocompetent syngeneic cancer models. Methods: eFT508 and its effect on mRNA translation, effector protein production, immune cell signaling and tumor infiltrating lymphocytes was evaluated in vitro using normal human T cells and in vivo utilizing immunocompetent syngeneic models. The mechanism of translational regulation of specific target genes was further evaluated in these model systems. Results: Dysregulated translation of messenger RNA (mRNA) plays a role in the pathogenesis of multiple solid tumors and hematological malignancies. MNK1 and MNK2 integrate signals from several oncogenic and immune signaling pathways (including RAS, p38 and toll-like receptors) by phosphorylating eukaryotic initiation factor 4E (eIF4E) and other key effector proteins including hnRNPA1 and PSF. Phosphorylation of these RNA-binding proteins by MNK1 and MNK2 selectively regulates the stability and translation of a subset of cellular mRNA that control tumor/stromal cell signaling and the tumor microenvironment. eFT508 inhibits both MNK1 and MNK2 through a reversible, ATP-competitive mechanism of action with an IC50 of 2 and 1 nM against MNK1 and MNK2 respectively. eFT508 is highly selective (≥100-fold) for MNK1 and MNK2 relative to over 400 other protein and lipid kinases. Ribosome profiling has demonstrated that inhibition of MNK1 and MNK2 by eFT508 selectively regulates the translational efficiency and mRNA stability of a subset of genes that include inflammatory cytokines/chemokines, regulators of reactive oxygen species (ROS), and effectors of anti-tumor immune response. Given the importance of both RAS signaling and translational control to immune cell function the immunological effect of eFT508 was evaluated in both normal human T cells in vitro and immunocompetent syngeneic cancer models in vivo. eFT508 treatment of normal donor T cells has no deleterious effect on CD3/CD28 activation of IL-2 production, T cell proliferation or on T cell viability. However, eFT508 selectively down regulates the induction of IL-10 and specific immune checkpoint mechanisms. The effect of eFT508 on IL-10 protein production corresponded with reduced mRNA stability. The in vivo antitumor effect of eFT508 was assessed in the CT26 BALB/C syngeneic tumor model. CT26 mouse tumor cell proliferation and survival are insensitive to eFT508 in vitro. In vivo, daily oral treatment with 1 mg/kg eFT508 results in significant anti-tumor activity and establishment of immune memory. In addition, combination of daily oral treatment of 1 mg/kg eFT508 with either anti-PD-1 or anti-PD-L1 monoclonal antibodies increases the number of responder animals and results in synergistic activity that corresponds to the modulation of tumor infiltrating lymphocyte populations. Conclusions: eFT508 is a selective, orally bioavailable small molecule inhibitor of MNK1 and MNK2 that can decrease the production of key immune checkpoint regulators and immunosuppressive cytokines. This novel mechanism of action triggers anti-tumor immune response in immunocompetent syngeneic animal models as a monotherapy and in combination with established immune checkpoint antibodies. eFT508 is currently under evaluation in two phase I clinical trials for patients with advanced solid tumors and patients with advanced lymphoma respectively. These findings support further clinical evaluation of eFT508 in combination with checkpoint blockade. This abstract is also being presented as Poster B29. Citation Format: Kevin R. Webster, Vikas K. Goel, Jocelyn Staunton, Ivy NJ Hung, Gregory S. Parker, Craig R. Stumpf, Jolene Molter, Gary G. Chiang, Christopher J. Wegerski, Samuel Sperry, Joan Chen, Vera Huang, Peggy A. Thompson, Chinh Tran, Justin T. Ernst, Stephen E. Webber, Paul A. Sprengeler, Siegfried H. Reich. eFT508: An oral, potent and highly selective inhibitor of MNK1 and MNK2, promotes anti-tumor immunity as a monotherapy and in combination with immune checkpoint blockade. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr PR11.

Dysregulated translation of messenger RNA (mRNA) plays a role in the pathogenesis of multiple solid tumors and hematological malignancies. MNK1 and MNK2 integrate signals from several oncogenic and immune signaling pathways (including RAS, Toll-like receptors and T cell receptor) by phosphorylating eukaryotic initiation factor 4E (eIF4E) and other key effector proteins including hnRNPA1 and PSF. Phosphorylation of these RNA-binding proteins by MNK1 and MNK2 selectively regulates the stability and translation of a subset of cellular mRNA that control tumor/stromal cell signaling, the tumor microenvironment and immune cell function. eFT508 is a potent and highly selective inhibitor of both MNK1 and MNK2. Ribosome profiling has demonstrated that inhibition of MNK1 and MNK2 by eFT508 selectively regulates the translational efficiency and mRNA stability of a subset of genes that include inflammatory cytokines/chemokines, regulators of stress response, and effectors of anti-tumor immune response. Given the importance of MAPK signaling and translational control to immune cell activation and differentiation, the immunological effect of eFT508 was further evaluated in both normal human immune cells in vitro and immunocompetent syngeneic cancer models in vivo. eFT508 treatment of normal donor T cells has no deleterious effect on αCD3/αCD28 stimulated IL-2 production, T cell proliferation or T cell viability. However, eFT508 selectively down regulates the induction of IL-10 and specific immune checkpoint receptors, including PD-1 and LAG3. Further evaluation of the mechanism of translational regulation has shown LAG3 mRNA contains specific sequence elements in the 5’-untranslated region (UTR) that confer sensitivity to eFT508. In addition, IL-10 mRNA is destabilized upon treatment with eFT508 leading to significant inhibition of IL-10 production in activated T cells. Furthermore, eFT508 treatment results in upregulation of MHC class II molecules on tumor cells, macrophage and dendritic cells through an IL-10/MARCH1 dependent mechanism. The in vivo antitumor effect of eFT508 was assessed in the CT26 BALB/C syngeneic tumor model. CT26 mouse tumor cell proliferation and survival are insensitive to eFT508 in vitro. In vivo, daily oral treatment with 1 mg/kg eFT508 results in significant anti-tumor activity, modulation of tumor infiltrating lymphocytes and establishment of immune memory. In addition, combination of eFT508 with either anti-PD-1 or anti-PD-L1 monoclonal antibodies results in marked efficacy, significantly increasing the percentage of responder animals. eFT508 is currently under evaluation in two phase I/II clinical trials for patients with advanced solid tumors and patients with advanced lymphoma respectively. These findings support further clinical evaluation of eFT508 in combination with checkpoint blockade. Citation Format: Kevin R. Webster, Vikas K. Goel, Jocelyn Staunton, Craig R. Stumpf, Rajesh Sharma, Ivy N. Hung, Gregory S. Parker, Jolene Molter, Gary G. Chiang, Christopher J. Wegerski, Samuel Sperry, Vera Huang, Joan Chen, Peggy A. Thompson, Chinh Tran, Justin T. Ernst, Paul A. Sprengeler, Siegfried H. Reich. eFT508, a potent and highly selective inhibitor of MNK1/2 regulates immune checkpoint and cytokine expression promoting anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 596. doi:10.1158/1538-7445.AM2017-596

Dysregulated translation of messenger RNA (mRNA) plays a role in the pathogenesis of multiple solid tumors and hematological malignancies. MNK1 and MNK2 integrate signals from several oncogenic and immune signaling pathways, including RAS, p38, and Toll-like receptor (TLR) pathways, by phosphorylating eukaryotic initiation factor 4E (eIF4E) and other key effector proteins including hnRNPA1 and PSF. Through phosphorylation of these regulatory proteins MNK1 and MNK2 selectively regulate the stability and translation of a subset of cellular mRNA. eFT508 is a potent, highly selective, and orally bioavailable MNK1 and MNK2 inhibitor. eFT508 has a half-maximal inhibitory concentration (IC50) of 1-2 nM against 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 a dose-dependent reduction in eIF4E phosphorylation at serine 209 (IC50 = 2-16 nM), consistent with previous findings that phosphorylation of this site is solely dependent upon MNK1/MNK2. In a panel of ~50 hematological cancers, eFT508 showed anti-proliferative activity against multiple DLBCL cell lines. Sensitivity to eFT508 in TMD8, OCI-Ly3 and HBL1 DLBCL cell lines was associated with dose-dependent decreases in production of pro-inflammatory cytokines including TNFα, IL-6, IL-10 and CXCL10. Further evaluation eFT508 mechanism of action demonstrated that decreased TNFα production correlated with a 2-fold decrease in TNFα mRNA half-life. These findings are consistent with MNK1 phosphorylation of specific RNA-binding proteins, eg, hnRNPA1, that regulate the stability and translation of mRNA containing specific AU-rich elements (ARE) in their 3'-untranslated regions (UTR). Pro-inflammatory cytokines are drivers of key hallmarks of cancer including tumor cell survival, migration and invasion, angiogenesis, and immune evasion, while also driving drug resistance. Therefore, eFT508 was tested in vivo in 7 subcutaneous human lymphoma xenograft models. Significant anti-tumor activity was observed in the TMD8 and HBL-1 ABC-DLBCL models, both of which harbor activating MyD88 mutations. In addition, eFT508 combined effectively with components of R-CHOP and with novel targeted agents, including ibrutinib and venetoclax, in human lymphoma models. These results underscore the potential of eFT508 for the treatment of DLBCL. eFT508 has also been characterized in nonclinical safety pharmacology and toxicology studies. Clinical trials in patients with hematological and other malignancies are planned.

A structure-based drug design strategy was used to optimize a novel benzolactam series of HSP90α/β inhibitors to achieve >1000-fold selectivity versus the HSP90 endoplasmic reticulum and mitochondrial isoforms (GRP94 and TRAP1, respectively). Selective HSP90α/β inhibitors were found to be equipotent to pan-HSP90 inhibitors in promoting the clearance of mutant huntingtin protein (mHtt) in vitro, however with less cellular toxicity. Improved tolerability profiles may enable the use of HSP90α/β selective inhibitors in treating chronic neurodegenerative indications such as Huntington's disease (HD). A potent, selective, orally available HSP90α/β inhibitor was identified (compound 31) that crosses the blood-brain barrier. Compound 31 demonstrated proof of concept by successfully reducing brain Htt levels following oral dosing in rats.