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Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signaling. Here, we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5′ untranslated regions, including those with multiple G-quadruplex elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules. Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC, including critical KRAS signaling molecules such as PI3K, RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox-related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC.Significance:These findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.

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Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signaling. Here, we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5′ untranslated regions, including those with multiple G-quadruplex elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules. Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC, including critical KRAS signaling molecules such as PI3K, RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox-related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC.Significance:These findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.

Simple Summary Our work establishes that amplification of 4EBP1, as a part of Chr. 8p11, creates a synthetic dependency on FGFR1 signaling in cancer. 4EBP1 is phosphorylated by FGFR1 and PI3K signaling, and accordingly cancer with 4EBP1-FGFR1 amplification is more sensitive to FGFR1 and PI3K inhibition due to inhibition of 4EBP1 phosphorylation. Moreover, we characterize the translational targets of 4EBP1 and identify that 4EBP1 specifically regulates the translation of genes involved in insulin signaling, glucose metabolism, and the inositol pathway that plays a role in cancer progression. The eIF4E translation initiation factor has oncogenic properties and concordantly, the inhibitory eIF4E-binding protein (4EBP1) is considered a tumor suppressor. The exact molecular effects of 4EBP1 activation in cancer are still unknown. Surprisingly, 4EBP1 is a target of genomic copy number gains (Chr. 8p11) in breast and lung cancer. We noticed that 4EBP1 gains are genetically linked to gains in neighboring genes, including WHSC1L1 and FGFR1. Our results show that FGFR1 gains act to attenuate the function of 4EBP1 via PI3K-mediated phosphorylation at Thr37/46, Ser65, and Thr70 sites. This implies that not 4EBP1 but instead FGFR1 is the genetic target of Chr. 8p11 gains in breast and lung cancer. Accordingly, these tumors show increased sensitivity to FGFR1 and PI3K inhibition, and this is a therapeutic vulnerability through restoring the tumor-suppressive function of 4EBP1. Ribosome profiling reveals genes involved in insulin signaling, glucose metabolism, and the inositol pathway to be the relevant translational targets of 4EBP1. These mRNAs are among the top 200 translation targets and are highly enriched for structure and sequence motifs in their 5 ' UTR, which depends on the 4EBP1-EIF4E activity. In summary, we identified the translational targets of 4EBP1-EIF4E that facilitate the tumor suppressor function of 4EBP1 in cancer.

Singh et al. show that MYC affects mRNA translation efficiency and start-site selection. Notable examples include the translation of mRNAs encoding most proteins of the electron transport chain and aberrant translation initiation site usage in the CD19 receptor that allows escape from CD19-directed CAR-T therapy., The oncogenic c-MYC (MYC) transcription factor has broad effects on gene expression and cell behavior. We show that MYC alters the efficiency and quality of mRNA translation into functional proteins. Specifically, MYC drives the translation of most protein components of the electron transport chain in lymphoma cells, and many of these effects are independent from proliferation. Specific interactions of MYC-sensitive RNA-binding proteins (e.g., SRSF1/RBM42) with 5′UTR sequence motifs mediate many of these changes. Moreover, we observe a striking shift in translation initiation site usage. For example, in low-MYC conditions, lymphoma cells initiate translation of the CD19 mRNA from a site in exon 5. This results in the truncation of all extracellular CD19 domains and facilitates escape from CD19-directed CAR-T cell therapy. Together, our findings reveal MYC effects on the translation of key metabolic enzymes and immune receptors in lymphoma cells., Graphical Abstract

Cancer cells adapt their metabolic activities to support growth and proliferation. However, increased activity of metabolic enzymes is not usually considered an initiating event in the malignant process. Here, we investigate the possible role of the enzyme serine hydroxymethyltransferase-2 (SHMT2) in lymphoma initiation. SHMT2 localizes to the most frequent region of copy number gains at chromosome 12q14.1 in lymphoma. Elevated expression of SHMT2 cooperates with BCL2 in lymphoma development; loss or inhibition of SHMT2 impairs lymphoma cell survival. SHMT2 catalyzes the conversion of serine to glycine and produces an activated one-carbon unit that can be used to support S-adenosyl methionine synthesis. SHMT2 induces changes in DNA and histone methylation patterns leading to promoter silencing of previously uncharacterized mutational genes, such as SASH1 and PTPRM. Together, our findings reveal that amplification of SHMT2 in cooperation with BCL2 is sufficient in the initiation of lymphomagenesis through epigenetic tumor suppressor silencing. Parsa et al. report a mechanism of lymphoma initiation involving cooperation of BCL2 and increased activity of the metabolic enzyme SHMT2, which imparts changes in DNA and histone methylation.

Simple Summary eIF4A-targeted translational inhibitors, such as silvestrol and its analogues, have emerged as strong anticancer therapies. Here, we tested the efficacy of eIF4A inhibition across a large and diverse panel of cancer cell lines and found B cell lymphomas to be the most sensitive group. Moreover, we performed a genetic screen and identified NRF2 activation as a major mechanism of resistance to silvestrol and related eIF4A inhibitors. Mechanistically, NRF2 activation broadly increases protein synthesis, and this effect is more pronounced on specific mRNAs that require eIF4A for translation. Finally, blocking NRF2 function by preventing its deglycation restores silvestrol sensitivity in cells that harbor NRF2 activation. Overall, our findings indicate that eIF4A inhibitors are a feasible therapeutic option against lymphoma and other cancers and that NRF2 activation status may be an important predictor of their efficacy. Abstract Inhibition of the eIF4A RNA helicase with silvestrol and related compounds is emerging as a powerful anti-cancer strategy. We find that a synthetic silvestrol analogue (CR-1-31 B) has nanomolar activity across many cancer cell lines. It is especially active against aggressive MYC+/BCL2+ B cell lymphomas and this likely reflects the eIF4A-dependent translation of both MYC and BCL2. We performed a genome-wide CRISPR/Cas9 screen and identified mechanisms of resistance to this new class of therapeutics. We identify three negative NRF2 regulators (KEAP1, CUL3, CAND1) whose inactivation is sufficient to cause CR1-31-B resistance. NRF2 is known to alter the oxidation state of translation factors and cause a broad increase in protein production. We find that NRF2 activation particularly increases the translation of some eIF4A-dependent mRNAs and restores MYC and BCL2 production. We know that NRF2 functions depend on removal of sugar adducts by the frutosamine-3-kinase (FN3K). Accordingly, loss of FN3K results in NRF2 hyper-glycation and inactivation and resensitizes cancer cells to eIF4A inhibition. Together, our findings implicate NRF2 in the translation of eIF4A-dependent mRNAs and point to FN3K inhibition as a new strategy to block NRF2 functions in cancer.

G-quadruplex (GQ) elements are conserved translational control elements that induce a requirement for RNA helicase activity for efficient mRNA translation. Computational structure analyses identify multiple GQ elements in the 5'UTRs of mRNAs encoding KRAS and key downstream signalling molecules. KRAS is a key oncogene in pancreatic ductal adenocarcinoma (PDAC) and many other cancers and except for the specific G12C KRAS mutation we do not have pharmacological inhibitors. We find that KRAS and downstream signalling molecules including PI3K, RALA, RAC2, MYC, MET, and YAP1 strictly depend on the RNA helicase eIF4A for their translation. This implies a potential utility for eIF4A/DDX2 inhibitors. CR31B is a silvestrol analogue that efficiently and selectively blocks eIF4A. CR31B kills PDAC cells at nanomolar concentrations and has significant single agent in vivo efficacy across xenograft, primary PDX, and murine models of PDAC. Together, our findings reveal coordinate, GQ- and eIF4A-mediated control of the translation of key RAS signalling molecules as a vulnerability in KRAS-driven pancreatic cancer. Citation Format: Kamini Singh, Jianan Lin, Nicolas Lecomte, Prathibha Mohan, Steve D. Leach, Zhengqing Ouyang, Hans-Guido Wendel. Coordinate translational control of the kras signaling pathway [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 1824.

New and effective therapeutics are urgently needed for the treatment of pancreatic ductal adenocarcinoma (PDAC). The eIF4A/DDX2 RNA helicase drives translation of mRNAs with highly structured 5′UTRs. The natural compound silvestrol and synthetic analogues are potent and selective inhibitors of eIF4A1/2 that show promising activity in models of hematologic malignancies. Here, we show silvestrol analogues have nanomolar activity against PDAC cell lines and organoids in vitro. Moreover, we see single-agent activity in the KRAS/p53 mouse PDAC model and also against PDAC xenograft and primary, patient-derived PDAC tumors. These therapeutic effects occur at nontoxic dose levels. Transcriptome-wide ribosome profiling, analyses of protein and gene expression, and translation reporter studies reveal that KRAS and the RAS signaling network is co-regulated by translation in an eIF4A dependent manner. eIF4A inhibitors block an oncogenic translation program in PDAC cells that includes G-quadruplex containing mRNAs such as KRAS, MYC, YAP1, MET, SMAD3, TGFβ and other components of the RAS signaling network. Together, our data indicate that pharmacologic inhibition of eIF4A disrupts oncoproteins production and shows efficacy across several PDAC models. Citation Format: Kamini Singh, Jianan Lin, Nicolas Lecomte, Prathibha Mohan, Askan Gokce, Viraj Sanghvi, Grbovic-Huezo Olivera, Man Jiang, Antonija Burčul, Stefan Stark, Agnes Viale, Paul B. Romesser, Elisa Destanchia, Rachel Bagni, Gunnar Rätsch, Steve D. Leach, Zhengqing Ouyang, Hans-Guido Wendel. KRAS and RAS signaling network is co-regulated and can be therapeutically blocked by targeting eIF4A dependent translation program [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B44.

Summary The NRF2 transcription factor controls a cell stress program that is implicated in cancer and there is great interest in targeting NRF2 for therapy. We show that NRF2 activity depends on Fructosamine-3-kinase (FN3K)—a kinase that triggers protein de-glycation. In its absence, NRF2 is extensively glycated, unstable, and defective at binding to small MAF proteins and transcriptional activation. Moreover, the development of hepatocellular carcinoma triggered by MYC and Keap1 inactivation depends on FN3K in vivo. N-acetyl cysteine treatment partially rescues the effects of FN3K loss on NRF2 driven tumor phenotypes indicating a key role for NRF2-mediated redox balance. Mass spectrometry reveals that other proteins undergo FN3K-sensitive glycation, including translation factors, heat shock proteins, and histones. How glycation affects their functions remains to be defined. In summary, our study reveals a surprising role for the glycation of cellular proteins and implicates FN3K as targetable modulator of NRF2 activity in cancer.

Pancreatic cancer is one of the most aggressive cancers with no targeted therapy available. RNA translation is activated in aggressive pancreatic cancer and at the same time is also refractory to mTor inhibition. We have used a translation inhibitor for eIF4A, RNA helicase that is downstream of mTOR signaling and can be functionally targeted in pancreatic cancer. We establish that Silvestrol and its analog CR-31B showed potent anti-tumor activity in pancreatic cancer cell lines in vitro and in vivo. Silvestrol/CR-31B reduced pancreatic cancer cells and organoids growth derived from mouse model of pancreatic cancer and human patient samples in vitro. Further we identify the genome wide translational targets of Silvestrol in pancreatic cancer cell line that lacks response to mTOR signaling through loss of EIF4EBP1. Silvestrol down regulate translation of many key oncogenes and others cellular factors involved in oncogenic signaling in pancreatic cancer. Silvestrol targets were also enriched for G-quadruplex structure in their 5’UTR. With this study we establish a new mechanism of targeting pancreatic cancer cells through translation inhibition and identify more proteins as therapeutic targets that are regulated independent of mTOR signaling. Citation Format: Kamini Singh, Stefan G. Stark, Agnes Viale, Prathibha Mohan, Man Jiang, Gunnar Rätsch, Hans-Guido Wendel.{Authors}. Targeting eIF4A dependent translation as therapeutics in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A56.