eIF4A Inhibitor Research Articles

Phase 1/2 dose expansion study evaluating first-in-class eIF4A inhibitor zotatifin in patients with ER+ metastatic breast cancer.

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 .

The flavagline FL3 interferes with the association of Annexin A2 with the eIF4F initiation complex and transiently stimulates the translation of annexin A2 mRNA.

Introduction: Annexin A2 (AnxA2) plays a critical role in cell transformation, immune response, and resistance to cancer therapy. Besides functioning as a calcium- and lipidbinding protein, AnxA2 also acts as an mRNA-binding protein, for instance, by interacting with regulatory regions of specific cytoskeleton-associated mRNAs. Methods and Results: Nanomolar concentrations of FL3, an inhibitor of the translation factor eIF4A, transiently increases the expression of AnxA2 in PC12 cells and stimulates shortterm transcription/translation of anxA2 mRNA in the rabbit reticulocyte lysate. AnxA2 regulates the translation of its cognate mRNA by a feed-back mechanism, which can partly be relieved by FL3. Results obtained using the holdup chromatographic retention assay results suggest that AnxA2 interacts transiently with eIF4E (possibly eIF4G) and PABP in an RNA-independent manner while cap pulldown experiments indicate a more stable RNA-dependent interaction. Short-term (2 h) treatment of PC12 cells with FL3 increases the amount of eIF4A in cap pulldown complexes of total lysates, but not of the cytoskeletal fraction. AnxA2 is only present in cap analogue-purified initiation complexes from the cytoskeletal fraction and not total lysates confirming that AnxA2 binds to a specific subpopulation of mRNAs. Discussion: Thus, AnxA2 interacts with PABP1 and subunits of the initiation complex eIF4F, explaining its inhibitory effect on translation by preventing the formation of the full eIF4F complex. This interaction appears to be modulated by FL3. These novel findings shed light on the regulation of translation by AnxA2 and contribute to a better understanding of the mechanism of action of eIF4A inhibitors.

Abstract LB353: The switch from cap-dependent to cap-independent translation reshapes the immunopeptidome of lung cancer

Abstract Cancer immunosurveillance relies on the presentation of peptide antigens on human leuckocyte antigen class I (HLA-I). HLA-I molecules present a diverse array of antigens, known as the “immunopeptidome”, which are recognized by cytotoxic CD8+ T cells. Recent evidence suggests that cryptic peptide antigens derived from non-canonical open reading frames (nuORFs) contribute to the immunopeptidome and drive CD8+ T cell responses. Integrating ribosome profiling (Ribo-seq) with mass spectrometry (MS) analyses has enabled the identification of several cryptic peptides derived from nuORFs including from putative non-coding regions including the 5’UTR, 3’UTR, long non coding RNAs (lncRNAs) and retained introns. Notably, many nuORFs undergo cap-independent translation. Various physiological and pharmacological stressors arrest canonical cap-dependent translation initiation and induce cap-independent translation. However, how this shift in translational dynamics influences the immunopeptidome is unknown. Using lung cancer cell lines, we show that the eIF4A inhibitor silvestrol induces cap-independent translation and reshapes the landscape of nuORF-derived peptide antigens. Silvestrol induced changes in the immunopeptidome strongly contrast with the global translation inhibitor homoharringtonine, which dampens antigen presentation. These observations suggest that the presentation of nuORF antigens in lung cancer is dynamic and tightly controlled by translational dynamics. More broadly, our study provokes reconsideration of translation inhibitors as immunomodulatory drugs that can change the HLA-I antigen repertoire and generate peptides that can act as new targets for cancer immunotherapy. Citation Format: Anika N. Ali, Andrew D. Weeden, Alex M. Jaeger. The switch from cap-dependent to cap-independent translation reshapes the immunopeptidome of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB353.

Abstract 4167: Translational control of T cell exhaustion in melanoma, perspectives in therapeutics

Abstract Background: Eukaryotic initiation factor 4F (eIF4F) complex plays a pivotal role in the selective translational control of protein expression in cancer. We previously showed that the inhibition of eIF4F had both a direct antitumor effect that could overcome resistance to BRAF inhibition in BRAFV600 melanoma and an indirect immune-mediated antitumor effect via PD-L1 down regulation resulting from STAT1 decreased translation. We now explore further the translational control of immune checkpoints (ICs) on T cell and its link with lymphocyte exhaustion. Methods: We performed polysome profiling (PP) on peripheral blood-derived activated and resting T lymphocytes as well as tumor-infiltrating lymphocytes (TILs) isolated from melanoma specimens in the presence or absence of silvestrol as an eIF4A (the helicase component of eIF4F) inhibitor. We studied the expression of ICs and exhaustion-associated transcription factors on lymphocytes at the various stages of exhaustion. The tumor cell lysing capacity of PBMC was evaluated with and without silvestrol. We also assessed the effect of silvestrol on tumor microenvironment in the BP syngeneic melanoma tumor model. Results: Silvestrol decreased the translation of multiple ICs in activated CD4+ and CD8+ T cells, including TOX, NFAT1, NFAT2, LAG3, and TIM3 (the two latter being also transcriptionally regulated). FACS analysis confirmed the dose-dependent decrease of protein expression of exhaustion-associated transcription factors TOX, NFATc1, TCF1 and T-bet in activated lymphocytes. This effect was more prominent in more exhausted T cells. Silvestrol decreased T cell proliferation but, in co-culture experiments, it increased immune cell killing of melanoma cells by T-lymphocytes. In the BP murine model, the antitumor effect of silvestrol was associated with denser T cell infiltration and less exhausted TILs than control tumors. Conclusion: Our findings demonstrate the role of eIF4F in the orchestrated regulation of functionally related proteins involved in T cell exhaustion. They highlight the drugability of eIF4A for the cancer treatment and suggest that eIF4A inhibitors could be useful with IC inhibitors in combination or sequentially. Citation Format: Samad Muhammadnejad, Biswendu Biswas, Ramdane Guemiri, Naima Benannoune, Jérémy Lavigne, Virginie Quidville, Jean-Yves Scoazec, Stephan Vagner, Caroline Robert. Translational control of T cell exhaustion in melanoma, perspectives in therapeutics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4167.

Abstract 4878: Transcriptional response to rocaglate translation inhibitors in lymphoma shows cellular adaptation through induction of stress and survival pathways

Abstract Background: 40% of diffuse large B-cell lymphoma (DLBCL) patients do not achieve cure in response to the standard immunochemotherapy. Relapsed and refractory tumors respond poorly to targeted signaling inhibitors due to pathway redundancies in this genetically heterogenous disease. Pharmacologic disruption of the cap-initiation complex eIF4F is an emerging clinical treatment strategy to bypass resistance. In preclinical experiments, B-lymphomas show high susceptibility to rocaglates, natural and synthetic cap-dependent translation inhibitors. Among these compounds, eFT226 (zotatifin), is currently in phase I/II clinical evaluation for solid tumors. Rocaglate mechanisms remain incompletely defined for use in B-cell lymphoma. Recent findings in our laboratory revealed translationally mediated adaptation that increased synthesis of many targets. Proteins (i.e.CD98hc) that paradoxically increase in production contribute to persistence of cells that survive treatment. By contrast, transcriptional responses to rocaglates remain minimally studied. Here we sought to fill this gap through unbiased transcriptomics. Method: RNA sequencing (RNA-seq) was performed on zotatifin treated DLBCL cells (SU-DHL10) vs DMSO controls. Genes with a false discovery rate p-value (FDR) ≤0.05, logarithm of counts per million reads (logCPM) ≥ 0 and P value ≤0.05 were considered differentially expressed. Log2 fold change (FC) ≥ +/-2-fold for a specific gene was considered significantly differentially expressed between the two groups. Pathway analysis was performed using Gene Set Enrichment Analysis (GSEA). Results: We found 4450 differentially expressed genes, 12 significantly upregulated, and 42 significantly downregulated in response to zotatifin (FC ≥ 2). Further pathways analyses showed 4/25 gene sets significantly upregulated, and 3/22 gene sets significantly downregulated (FDR <0.25). We found significantly upregulated genes related to mechanisms stress and survival pathways including NF-kB induction by TNF signaling (normalized enriched score (NES)=2.28; p-value <0.0001), genes induced in response to hypoxia translational stress responses (NES=1.85; nominal p-value=0.008). Conclusions: These results implicate induction of specific survival pathways that induce transcriptionally mediated adaptation to the stress of rocaglate therapy, but it is unclear if these responses can result in altered protein production in the context of eIF4A inhibition. Parallel assessment of the translatome currently under analysis will provide a more complete picture of overall adaptation and provide rational combination targets for rocaglates in lymphoma as clinical development progresses. Citation Format: Paola Manara, Tyler Andrew Cunningham, Jr Jyun David Ho, Olivia B. Lightfuss, Abdessamad Youssfi Alaoui, Jonathan Harry Schatz. Transcriptional response to rocaglate translation inhibitors in lymphoma shows cellular adaptation through induction of stress and survival pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4878.

Rapamycin-Induced Feedback Activation of eIF4E-EIF4A Dependent mRNA Translation in Pancreatic Cancer.

Pancreatic cancer cells adapt molecular mechanisms to activate the protein synthesis to support tumor growth. This study reports the mTOR inhibitor rapamycin's specific and genome-wide effect on mRNA translation. Using ribosome footprinting in pancreatic cancer cells that lack the expression of 4EBP1, we establish the effect of mTOR-S6-dependent mRNAs translation. Rapamycin inhibits the translation of a subset of mRNAs including p70-S6K and proteins involved in the cell cycle and cancer cell growth. In addition, we identify translation programs that are activated following mTOR inhibition. Interestingly, rapamycin treatment results in the translational activation of kinases that are involved in mTOR signaling such as p90-RSK1. We further show that phospho-AKT1 and phospho-eIF4E are upregulated following mTOR inhibition suggesting a feedback activation of translation by rapamycin. Next, targeting eIF4E and eIF4A-dependent translation by using specific eIF4A inhibitors in combination with rapamycin shows significant growth inhibition in pancreatic cancer cells. In short, we establish the specific effect of mTOR-S6 on translation in cells lacking 4EBP1 and show that mTOR inhibition leads to feedback activation of translation via AKT-RSK1-eIF4E signals. Therefore, targeting translation downstream of mTOR presents a more efficient therapeutic strategy in pancreatic cancer.

Oncogenic PKA signaling increases c-MYC protein expression through multiple targetable mechanisms.

Genetic alterations that activate protein kinase A (PKA) are found in many tumor types. Yet, their downstream oncogenic signaling mechanisms are poorly understood. We used global phosphoproteomics and kinase activity profiling to map conserved signaling outputs driven by a range of genetic changes that activate PKA in human cancer. Two signaling networks were identified downstream of PKA: RAS/MAPK components and an Aurora Kinase A (AURKA)/glycogen synthase kinase (GSK3) sub-network with activity toward MYC oncoproteins. Findings were validated in two PKA-dependent cancer models: a novel, patient-derived fibrolamellar carcinoma (FLC) line that expresses a DNAJ-PKAc fusion and a PKA-addicted melanoma model with a mutant type I PKA regulatory subunit. We identify PKA signals that can influence both de novo translation and stability of the proto-oncogene c-MYC. However, the primary mechanism of PKA effects on MYC in our cell models was translation and could be blocked with the eIF4A inhibitor zotatifin. This compound dramatically reduced c-MYC expression and inhibited FLC cell line growth in vitro. Thus, targeting PKA effects on translation is a potential treatment strategy for FLC and other PKA-driven cancers.

EIF4A1 Is a Prognostic Marker and Actionable Target in Human Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a primary liver tumor with high lethality and increasing incidence worldwide. While tumor resection or liver transplantation is effective in the early stages of the disease, the therapeutic options for advanced HCC remain limited and the benefits are temporary. Thus, novel therapeutic targets and more efficacious treatments against this deadly cancer are urgently needed. Here, we investigated the pathogenetic and therapeutic role of eukaryotic initiation factor 4A1 (eIF4A1) in this tumor type. We observed consistent eIF4A1 upregulation in HCC lesions compared with non-tumorous surrounding liver tissues. In addition, eIF4A1 levels were negatively correlated with the prognosis of HCC patients. In HCC lines, the exposure to various eIF4A inhibitors triggered a remarkable decline in proliferation and augmented apoptosis, paralleled by the inhibition of several oncogenic pathways. Significantly, anti-growth effects were achieved at nanomolar concentrations of the eIF4A1 inhibitors and were further increased by the simultaneous administration of the pan mTOR inhibitor, Rapalink-1. In conclusion, our results highlight the pathogenetic relevance of eIF4A1 in HCC and recommend further evaluation of the potential usefulness of pharmacological combinations based on eIF4A and mTOR inhibitors in treating this aggressive tumor.

Regulatory T cells suppress CD4+ effector T cell activation by controlling protein synthesis.

Regulatory T cells (Tregs) suppress the activation and subsequent effector functions of CD4 effector T cells (Teffs). However, molecular mechanisms that enforce Treg-mediated suppression in CD4 Teff are unclear. We found that Tregs suppressed activation-induced global protein synthesis in CD4 Teffs prior to cell division. We analyzed genome-wide changes in the transcriptome and translatome of activated CD4 Teffs. We show that mRNAs encoding for the protein synthesis machinery are regulated at the level of translation in activated CD4 Teffs by Tregs. Tregs suppressed global protein synthesis of CD4 Teffs by specifically inhibiting mRNAs of the translation machinery at the level of mTORC1-mediated translation control through concerted action of immunosuppressive cytokines IL-10 and TGFβ. Lastly, we found that the therapeutic targeting of protein synthesis with the RNA helicase eIF4A inhibitor rocaglamide A can alleviate inflammatory CD4 Teff activation caused by acute Treg depletion in vivo. These data show that peripheral tolerance is enforced by Tregs through mRNA translational control in CD4 Teffs.

EIF4A inhibition targets bioenergetic homeostasis in AML MOLM-14 cells in vitro and in vivo and synergizes with cytarabine and venetoclax.

Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. A promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1). Clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. Here, we explored a strategy to curtail these drawbacks through inhibition of an importanteffector of themTORC1signaling pathway, the eukaryotic initiation factor 4A (eIF4A). We tested the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, in combination with cytosine arabinoside (araC) or the BCL2 inhibitor venetoclax. We utilized the MOLM-14 human AML cell line to model chemoresistant disease both in vitro and in vivo. In eIF4Ai-treated cells, we assessed for changes in survival, apoptotic priming, de novo protein synthesis, targeted intracellular metabolite content, bioenergetic profile, mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP). eIF4Ai exhibits anti-leukemia activity in vivo while sparing non-malignant myeloid cells. In vitro, eIF4Ai synergizes with two therapeutic agents in AML, araC and venetoclax. EIF4Ai reduces mitochondrial membrane potential (MMP) and the rate of ATP synthesis from mitochondrial respiration and glycolysis. Furthermore, eIF4i enhanced apoptotic priming while reducing the expression levels of the antiapoptotic factors BCL2, BCL-XL and MCL1. Concomitantly, eIF4Ai decreases intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mtROS. In vitro redox stress contributes to eIF4Ai cytotoxicity, as treatment with a ROS scavenger partially rescued the viability of eIF4A inhibition. We discovered that chemoresistant MOLM-14 cells rely on eIF4A-dependent cap translation for survival in vitro and in vivo. EIF4A drives an intrinsic metabolic program sustaining bioenergetic and redox homeostasis and regulates the expression of anti-apoptotic proteins. Overall, our work suggests that eIF4A-dependent cap translation contributes to adaptive processes involved in resistance to relevant therapeutic agents in AML.

Enzymatic and Molecular Characterization of Anti-Leishmania Molecules That Differently Target Leishmania and Mammalian eIF4A Proteins, LieIF4A and eIF4AMus.

Previous investigations of the Leishmania infantum eIF4A-like protein (LieIF4A) as a potential drug target delivered cholestanol derivatives inhibitors. Here, we investigated the mode of action of cholesterol derivatives as a novel scaffold structure of LieIF4A inhibitors on the RNA-dependent ATPase activity of LieIF4A and its mammalian ortholog (eIF4AI). We compared their biochemical effects on RNA-dependent ATPase activities of both proteins and investigated if rocaglamide, a known inhibitor of eIF4A, could affect LieIF4A as well. Kinetic measurements were conducted at different concentrations of ATP, of the compound and in the presence of saturating whole yeast RNA concentrations. Kinetic analyses showed different ATP binding affinities for the two enzymes as well as different sensitivities to 7-α-aminocholesterol and rocaglamide. The 7-α-aminocholesterol inhibited LieIF4A with a higher binding affinity relative to cholestanol analogs. Cholesterol, another tested sterol, had no effect on the ATPase activity of LieIF4A or eIF4AI. The 7-α-aminocholesterol demonstrated an anti-Leishmania activity on L. infantum promastigotes. Additionally, docking simulations explained the importance of the double bond between C5 and C6 in 7-α-aminocholesterol and the amino group in the C7 position. In conclusion, Leishmania and mammalian eIF4A proteins appeared to interact differently with effectors, thus making LieIF4A a potential drug against leishmaniases.

Novel eIF4A1 inhibitors with anti‐tumor activity in lymphoma

BackgroundDeregulated translation initiation is implicated extensively in cancer initiation and progression. It is actively pursued as a viable target that circumvents the dependency on oncogenic signaling, a significant factor in current strategies. Eukaryotic translation initiation factor (eIF) 4A plays an essential role in translation initiation by unwinding the secondary structure of messenger RNA (mRNA) upstream of the start codon, enabling active ribosomal recruitment on the downstream genes. Several natural product molecules with similar scaffolds, such as Rocaglamide A (RocA), targeting eIF4A have been reported in the last decade. However, their clinical utilization is still elusive due to several pharmacological limitations. In this study we identified new eIF4A1 inhibitors and their possible mechanisms.MethodsIn this report, we conducted a pharmacophore-based virtual screen of RocA complexed with eIF4A and a polypurine RNA strand for novel eIF4A inhibitors from commercially available compounds in the MolPort Database. We performed target-based screening and optimization of active pharmacophores. We assessed the effects of novel compounds on biochemical and cell-based assays for efficacy and mechanistic evaluation.ResultsWe validated three new potent eIF4A inhibitors, RBF197, RBF 203, and RBF 208, which decreased diffuse large B-cell lymphoma (DLBCL) cell viability. Biochemical and cellular studies, molecular docking, and functional assays revealed that thosenovel compounds clamp eIF4A into mRNA in an ATP-independent manner. Moreover, we found that RBF197 and RBF208 significantly depressed eIF4A-dependent oncogene expression as well as the colony formation capacity of DLBCL. Interestingly, exposure of these compounds to non-malignant cells had only minimal impact on their growth and viability.ConclusionsIdentified compounds suggest a new strategy for designing novel eIF4A inhibitors.

Inhibition of the Translation Initiation Factor eIF4A Enhances Tumor Cell Radiosensitivity.

A fundamental component of cellular radioresponse is the translational control of gene expression. Because a critical regulator of translational control is the eukaryotic translation initiation factor 4F (eIF4F) cap binding complex, we investigated whether eIF4A, the RNA helicase component of eIF4F, can serve as a target for radiosensitization. Knockdown of eIF4A using siRNA reduced translational efficiency, as determined from polysome profiles, and enhanced tumor cell radiosensitivity as determined by clonogenic survival. The increased radiosensitivity was accompanied by a delayed dispersion of radiation-induced γH2AX foci, suggestive of an inhibition of DNA double-strand break repair. Studies were then extended to (-)-SDS-1-021, a pharmacologic inhibitor of eIF4A. Treatment of cells with the rocaglate (-)-SDS-1-021 resulted in a decrease in translational efficiency as well as protein synthesis. (-)-SDS-1-021 treatment also enhanced the radiosensitivity of tumor cell lines. This (-)-SDS-1-021-induced radiosensitization was accompanied by a delay in radiation-induced γH2AX foci dispersal, consistent with a causative role for the inhibition of double-strand break repair. In contrast, although (-)-SDS-1-021 inhibited translation and protein synthesis in a normal fibroblast cell line, it had no effect on radiosensitivity of normal cells. Subcutaneous xenografts were then used to evaluate the in vivo response to (-)-SDS-1-021 and radiation. Treatment of mice bearing subcutaneous xenografts with (-)-SDS-1-021 decreased tumor translational efficiency as determined by polysome profiles. Although (-)-SDS-1-021 treatment alone had no effect on tumor growth, it significantly enhanced the radiation-induced growth delay. These results suggest that eIF4A is a tumor-selective target for radiosensitization.

Identification of structurally re-engineered rocaglates as inhibitors against hepatitis E virus replication

Hepatitis E virus (HEV) infections are a leading cause of acute viral hepatitis in humans and pose a considerable threat to public health. Current standard of care treatment is limited to the off-label use of nucleoside-analog ribavirin (RBV) and PEGylated interferon-α, both of which are associated with significant side effects and provide limited efficacy.In the past few years, a promising natural product compound class of eukaryotic initiation factor 4A (eIF4A) inhibitors (translation initiation inhibitors), called rocaglates, were identified as antiviral agents against RNA virus infections. In the present study, we evaluated a total of 205 synthetic rocaglate derivatives from the BU-CMD compound library for their antiviral properties against HEV. At least eleven compounds showed inhibitory activities against the HEV genotype 3 (HEV-3) subgenomic replicon below 30 nM (EC50 value) as determined by Gaussia luciferase assay. Three amidino-rocaglates (ADRs) (CMLD012073, CMLD012118, and CMLD012612) possessed antiviral activity against HEV with EC50 values between 1 and 9 nM. In addition, these three selected compounds inhibited subgenomic replicons of different genotypes (HEV-1 [Sar55], wild boar HEV-3 [83-2] and human HEV-3 [p6]) in a dose-dependent manner and at low nanomolar concentrations. Furthermore, tested ADRs tend to be better tolerated in primary hepatocytes than hepatoma cancer cell lines and combination treatment of CMLD012118 with RBV and interferon-α (IFN-α) showed that CMLD012118 acts additive to RBV and IFN-α treatment. In conclusion, our results indicate that ADRs, especially CMLD012073, CMLD012118, and CMLD012612 may prove to be potential therapeutic candidates for the treatment of HEV infections and may contribute to the discovery of pan-genotypic inhibitors in the future.

Abstract 835: Estrogen receptor expression and ER dependent breast tumor growth are dependent on translation initiation factor EIF4A

Abstract The majority of human breast cancers are dependent on Estrogen Receptor Alpha (ER) and sensitive to its inhibition. In advanced, ER+ dependent breast cancers, resistance usually develops and is associated with insensitivity of the estrogen receptor to inhibition. Mutations that activate PI3K signaling occur in over 40% of ER-driven breast cancers. The PI3K pathway regulates cap-dependent protein translation by controlling mTOR complex I (mTORC1). Inhibitors of PI3K/mTOR are effective in this setting when given with anti-estrogens, but induce ER activity and expression. We now show that despite reducing global cap-dependent translation, PI3K/mTOR inhibition does not reduce ER translation or expression. Translation of ER instead depends on the translation initiation factor, EIF4A. Inhibitors of EIF4A significantly reduce the expression of WT and mutant ER, with attendant blockage of breast cancer model growth in vivo, including models driven by estrogen-independent ER fusions that are unaffected by estrogen receptor antagonists. The utility of EIF4A inhibition can be enhanced when combined with Fulvestrant, a degrader of ER. Combining inhibition of ER translation and induction of ER degradation causes synergistic deep and durable inhibition of ER expression and tumor growth. Inhibition of ER translation represents a new potent strategy for treating ER-dependent breast cancers with acquired resistance to current therapies. Citation Format: Jacob A. Boyer, Madeline A. Dorso, Corina Amor, Jason Reiter, Jianing Xu, Elisa de Stanchina, Hans-Guido Wendel, Sarat Chandarlapaty, Neal Rosen. Estrogen receptor expression and ER dependent breast tumor growth are dependent on translation initiation factor EIF4A [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 835.

First-in-human phase 1/2 dose escalation and expansion study evaluating first-in-class eIF4A inhibitor zotatifin in patients with solid tumors.

3081 Background: Zotatifin (eFT226) is a first in class, potent and sequence selective inhibitor of RNA helicase eIF4A1 that promotes stable mRNA:eIF4A:drug ternary complex at specific polypurine motifs within the 5’-UTR, preventing ribosome docking and thus, efficient translation of select transcripts. In preclinical models zotatifin treatment simultaneously down-regulated translation of numerous oncogenes with complex 5’ mRNA structures, including ERBB2, FGFR1/2, EGFR, KRAS, and CCND1. Methods: Patients (pts) with select advanced solid tumors harboring mutations/amplification of ERBB2, FGFR1, FGFR2, EGFR, or KRAS or with pancreatic cancer were enrolled into 3+3 dose escalation portion of the protocol (Part 1), and indication specific expansions continue to enroll at recommended phase 2 dose (RP2D; Part 2). The primary endpoints of Part 1 include determination of safety, tolerability, and maximum tolerated dose (MTD)/RP2D; additional endpoints include characterization of pharmacokinetic, pharmacodynamic (including from blood-based assay during escalation and from pre- and on-treatment biopsy at/near MTD with reverse phase protein array (RPPA)), and initial efficacy. Results: As of cut-off date Jan 13, 2022, Dose escalation phase (Part 1) included 37 patients treated with zotatifin at dose levels: 0.005, 0.01, 0.02, 0.035 mg/Kg IV weekly, and 0.035, 0.05, 0.07, 0.1 mg/kg IV 2 weeks-on and 1 week-off. DLTs were observed in 3 patients: Gr 2 thrombocytopenia (0.035 mg/kg weekly), Gr 3 anemia (0.1 mg/kg) and Gr 3 gastrointestinal bleed resulting in anemia (0.1 mg/kg). MTD/RP2D is 0.07 mg/Kg IV 2 weeks-on and 1 week-off. The most common treatment emergent adverse events (TEAEs) in Part 1 include: fatigue, anemia, diarrhea and dyspnea. The most common AEs at RP2D (n = 16 pts; Part 1 and Part 2) include: anemia (25% all gr 1 or 2), fatigue (25% all gr 1 or 2) and dyspnea (19%; 13% Gr 3) diarrhea (13%, all Gr 1 or 2). Pharmacokinetics were generally linear and dose proportional and exposures at MTD/RP2D are consistent with target pharmacologic levels in preclinical models. Blood based biomarkers showed dose- and time- dependent evidence of target engagement. Pre- and on-treatment biopsy data in expansion patients showed decreased expression of target proteins. No patient in dose escalation experienced an objective tumor response; initial efficacy data from Part 2 and at RP2D will be presented. Conclusions: eIF4A inhibitor zotatifin achieves pharmacologically relevant exposures with on-target AEs that are manageable at the MTD, with evidence of target knockdown from on-treatment biopsies. Part 2 indication-specific expansions (including in ER+ FGFR-amplified MBC as single agent, ER+ MBC in combination with fulvestrant and abemaciclib, and KRAS NSCLC in combination with sotorasib) are on-going. Clinical trial information: NCT04092673.

The eIF4A Inhibitor Silvestrol Blocks the Growth of Human Glioblastoma Cells by Inhibiting AKT/mTOR and ERK1/2 Signaling Pathway

The most frequently identified central nervous system tumor in adults is glioblastoma multiforme (GBM). GBM prognosis remains poor despite multimodal treatment, i.e., surgery and radiation therapy with concurrent temozolomide-based chemotherapy. Silvestrol, an eIF4A inhibitor, has been demonstrated to be able to kill tumor cells in previous studies. In this study, it was found that silvestrol considerably attenuated the proliferative potential of U251 and U87 glioma cells and reduced expression of cyclin D1. In addition, silvestrol reduced the level of ERK1/2 and decreased the levels of AKT phosphorylation. Unfortunately, the effect of silvestrol in inhibiting GBM cells was greatly reduced with hypoxia, and the downregulation in AKT/mTOR and ERK1/2 were also rescued with an upregulation of HIF1α, which warranted further research. Taken together, silvestrol exerted antitumor effects in GBM cells by inhibiting the AKT/mTOR and ERK1/2 signaling cascades.

B-cell receptor signaling induces proteasomal degradation of PDCD4 via MEK1/2 and mTORC1 in malignant B cells

B-cell receptor (BCR) signaling plays a major role in the pathogenesis of B-cell malignancies and is an established target for therapy, including in chronic lymphocytic leukemia cells (CLL), the most common B-cell malignancy. We previously demonstrated that activation of BCR signaling in primary CLL cells downregulated expression of PDCD4, an inhibitor of the translational initiation factor eIF4A and a potential tumor suppressor in lymphoma. Regulation of the PDCD4/eIF4A axis appeared to be important for expression of the MYC oncoprotein as MYC mRNA translation was increased following BCR stimulation and MYC protein induction was repressed by pharmacological inhibition of eIF4A. Here we show that MYC expression is also associated with PDCD4 down-regulation in CLL cells in vivo and characterize the signaling pathways that mediate BCR-induced PDCD4 down-regulation in CLL and lymphoma cells. PDCD4 downregulation was mediated by proteasomal degradation as it was inhibited by proteasome inhibitors in both primary CLL cells and B-lymphoma cell lines. In lymphoma cells, PDCD4 degradation was predominantly dependent on signaling via the AKT pathway. By contrast, in CLL cells, both ERK and AKT pathways contributed to PDCD4 down-regulation and dual inhibition using ibrutinib with either MEK1/2 or mTORC1 inhibition was required to fully reverse PDCD4 down-regulation. Consistent with this, dual inhibition of BTK with MEK1/2 or mTORC1 resulted in the strongest inhibition of BCR-induced MYC expression. This study provides important new insight into the regulation of mRNA translation in B-cell malignancies and a rationale for combinations of kinase inhibitors to target translation control and MYC expression.

Differential Effects on the Translation of Immune-Related Alternatively Polyadenylated mRNAs in Melanoma and T Cells by eIF4A Inhibition.

Simple SummaryImmune checkpoints blockade has emerged as an effective approach to prevent immune escape of tumor cells, and constitutes a powerful anti-cancer therapeutic strategy. Regulation of the expression of genes encoding immune checkpoint inhibitors has thus become an increasingly important field of study. Beyond transcription, gene expression is regulated at several post-transcriptional levels including pre-mRNA 3′-end processing and mRNA translation. More specifically, the eIF4F translation initiation complex represents an important hub for oncogenic signaling in the etiology of different cancers. The eIF4A RNA helicase component of the eIF4F can be inhibited by the widely characterized small molecule inhibitor silvestrol. Here, we evaluated the effect of eIF4A inhibition with silvestrol on the translation of alternatively polyadenylated mRNAs in melanoma cell lines and activated T cells. We show that silvestrol can selectively inhibit the translation of alternatively polyadenylated isoforms of genes encoding key immune-related proteins.Targeting the translation initiation complex eIF4F, which binds the 5′ cap of mRNAs, is a promising anti-cancer approach. Silvestrol, a small molecule inhibitor of eIF4A, the RNA helicase component of eIF4F, inhibits the translation of the mRNA encoding the signal transducer and activator of transcription 1 (STAT1) transcription factor, which, in turn, reduces the transcription of the gene encoding one of the major immune checkpoint proteins, i.e., programmed death ligand-1 (PD-L1) in melanoma cells. A large proportion of human genes produce multiple mRNAs differing in their 3′-ends through the use of alternative polyadenylation (APA) sites, which, when located in alternative last exons, can generate protein isoforms, as in the STAT1 gene. Here, we provide evidence that the STAT1α, but not STAT1β protein isoform generated by APA, is required for silvestrol-dependent inhibition of PD-L1 expression in interferon-γ-treated melanoma cells. Using polysome profiling in activated T cells we find that, beyond STAT1, eIF4A inhibition downregulates the translation of some important immune-related mRNAs, such as the ones encoding TIM-3, LAG-3, IDO1, CD27 or CD137, but with little effect on the ones for BTLA and ADAR-1 and no effect on the ones encoding CTLA-4, PD-1 and CD40-L. We next apply RT-qPCR and 3′-seq (RNA-seq focused on mRNA 3′ ends) on polysomal RNAs to analyze in a high throughput manner the effect of eIF4A inhibition on the translation of APA isoforms. We identify about 150 genes, including TIM-3, LAG-3, AHNAK and SEMA4D, for which silvestrol differentially inhibits the translation of APA isoforms in T cells. It is therefore crucial to consider 3′-end mRNA heterogeneity in the understanding of the anti-tumor activities of eIF4A inhibitors.

Abstract P145: The RNA helicase EIF4A is a therapeutic vulnerability in triple-negative breast cancer

Abstract Background: Translational control of oncoprotein expression has been implicated in the pathogenesis of multiple solid tumors. The eukaryotic translation initiation factor 4A (eIF4A) is an RNA helicase that catalyzes the unwinding of secondary structure in the 5′-untranslated region (5′-UTR) of mRNAs to facilitate translation initiation. eIF4A is an essential gene in many cancer cell lines and multiple oncogenes or anti-apoptotic proteins that contain structured 5’-UTRs have been shown to require enhanced eIF4A activity for translation. Zotatifin (eFT226) is a selective eIF4A inhibitor that has been shown previously to inhibit tumor growth in several immunocompromised mouse models and is currently in Phase I clinical trials as a single agent (NCT04092673). In this study we investigated the effects of Zotatifin both as a single agent and in combination with standard-of-care chemotherapy on tumor progression in several syngeneic triple-negative breast cancer (TNBC) mouse models. Methods: The activity of Zotatifin was assessed as single agent in eight different syngeneic p53-null mouse TNBC models across several intrinsic molecular subtypes. Synergism of Zotatifin with chemotherapy was evaluated in three responsive models. In addition, a Zotatifin-resistant model was developed following prolonged drug treatment. Drug response mechanisms and the effect on the tumor immune microenvironment were determined by tandem mass tag mass spectrometry, reverse phase protein array (RPPA) and mass cytometry. Results: Zotatifin treatment slowed tumor growth in six out of eight syngeneic TNBC models without obvious toxicity. Mass spectrometry of acutely treated tumors revealed that Zotatifin inhibited proliferative and stem cell signaling pathways including E2F targets, G2/M checkpoints, and NOTCH signaling, and induced proteins involved in Interferon-α and Interferon-γ responses. Mass cytometry analyses showed that Zotatifin suppressed the infiltration of M2 macrophages and neutrophils and promoted the infiltration of conventional dendritic cells to the tumor microenvironment. Interestingly, combination therapy of Zotatifin with chemotherapy significantly slowed tumor growth and improved survival. However, drug resistance emerged after prolonged treatment. The resistant tumors strikingly displayed a mesenchymal histology and an increased activity of the PI3K-AKT-mTOR pathway as determined by RPPA analysis. Combination treatment of Zotatifin with the mTOR inhibitor Everolimus prolonged survival. Conclusions: eIF4A is a therapeutic vulnerability in TNBC. Zotatifin inhibits tumor progression both through tumor-intrinsic mechanisms and by targeting the tumor immune microenvironment. Synergism between Zotatifin and chemotherapy supports the clinical potential of combination therapies. Citation Format: Na Zhao, Jeffrey Mark Rosen. The RNA helicase EIF4A is a therapeutic vulnerability in triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P145.