Translation initiation factor eIF4F modifies the dexamethasone response in multiple myeloma Francis Roberta, William Romanb, Alexandre Bramoulléc, Christof Fellmannd, Anne Roulstonc, Chaim Shustikb, John A. Porco, Jr.e, Gordon C. Shorea,c, Michael Sebagb,1, and Jerry Pelletiera,c,f,1 Departments of aBiochemistry, bMedicine and Hematology, and fOncology, and cThe Rosalind and Morris Goodman Cancer Research Center, McGill University, Montreal, QC, Canada H3G 1Y6; dMirimus, Inc., Cold Spring Harbor, NY 11724; and eCenter for Chemical Methodology and Library Development, Boston University, Boston, MA 02215 Edited* by David E. Housman, Massachusetts Institute of Technology, Cambridge, MA, and approved August 5, 2014 (received for review February 24, 2014) Enhanced protein synthesis capacity is associated with increased PI3K/mTOR and MAPK pathways intimately impinge on the tumor cell survival, proliferation, and resistance to chemother- translation pathway, linking this energetically demanding process apy. Cancers like multiple myeloma (MM), which display elevated to intra- and extracellular proliferation and survival cues (4). As activity in key translation regulatory nodes, such as the PI3K/ well, overexpression of MYC, a master regulator of ribosome mammalian target of rapamycin and MYC-eukaryotic initiation biogenesis and translation, is frequently observed in MM (5–7) factor (eIF) 4E pathways, are predicted to be particularly sensitive and is associated with a poor prognosis (7, 8). Deregulated to therapeutic strategies that target this process. To identify novel translational control is therefore a central feature of MM, with vulnerabilities in MM, we undertook a focused RNAi screen in perturbations occurring at the level of core components and which components of the translation apparatus were targeted. regulatory pathways. Our screen was designed to identify synthetic lethal relationships One of the best-studied MYC effectors and a downstream between translation factors or regulators and dexamethasone target of the PI3K/mTOR pathway implicated in translational (DEX), a corticosteroid used as frontline therapy in this disease. We control is eIF4F (4, 5, 9). eIF4F is a heterotrimeric complex find that suppression of all three subunits of the eIF4F cap-binding consisting of: (i) eIF4E, a cap-binding protein; (ii) eIF4A, an complex synergizes with DEX in MM to induce cell death. Using RNA helicase implicated in remodeling mRNA structure; and GENETICS a suite of small molecules that target various activities of eIF4F, we (iii) eIF4G, a large scaffolding protein. The association of eIF4E observed that cell survival and DEX resistance are attenuated with the eIF4F complex is regulated by mTOR, with mTOR upon eIF4F inhibition in MM cell lines and primary human samples. activation leading to stimulation of eIF4F formation. Elevated Levels of MYC and myeloid cell leukemia 1, two known eIF4F- PI3K/mTOR signaling flux or increased MYC levels exert pro- responsive transcripts and key survival factors in MM, were found activating effects on cap-dependent translation, and con- reduced upon eIF4F inhibition, and their independent suppres- sequently on cellular proliferation and survival (10). sion also synergized with DEX. Inhibition of eIF4F in MM exerts The PI3K/mTOR axis is being probed as a therapeutic target pleotropic effects unraveling a unique therapeutic opportunity. in MM (11). MM cells are sensitive to mTOR inhibition by rapa- mycin-related molecules (12), but the presence of an mTOR- silvestrol | hippuristanol | eIF4A | RNAi screening S6K-insulin receptor substrate-1 negative-feedback loop, which reactivates PI3K and AKT upon mTOR inhibition, diminishes ultiple myeloma (MM) is a bone marrow-derived malig- the efficacy of rapalogs (13). Second-generation mTOR complex Mnancy of plasma cells that typically produce a monoclonal (mTORC) kinase inhibitors (KIs) (e.g., OSI-027) and dual-spec- immunoglobulin (Ig). It is the second most frequent hema- ificity PI3K/mTOR KIs [e.g., (NVP)-BEZ235] avoid PI3K/AKT tological neoplasm in adults, with ∼20,000 newly diagnosed cases annually in the United States. Corticosteroids [predni- Significance sone and dexamethasone (DEX)] are common to all treat- ment regimens, usually in combination with an alkylating Multiple myeloma (MM) is a cancer that develops in the bone agent (e.g., melphalan) or more recently introduced agents, marrow and remains incurable to this day. It is a cancer type such as proteasome inhibitors (bortezomib and carfilzomib) that shows hallmarks of deregulated protein synthesis control. and immunomodulatory agents (thalidomide, lenalidomide, and To uncover new vulnerabilities in this disease, we performed pomalidomide). Despite the single-agent activity of these novel a focused RNAi screen to identify components of the trans- treatment options, enhanced clinical activity is observed when lation apparatus that, when depleted, would sensitize tumor they are used in combination with DEX. cells to dexamethasone (DEX), a component of frontline ther- The MM genomic landscape includes recurrent DNA trans- apy in this cancer. We found that suppression of eukaryotic locations involving mostly the IgH locus, chromosomal gains and initiation factor 4F, a heterotrimeric complex required for cap- losses, and a significant number of mutations in genes involved in dependent translation initiation, is a modifier of the DEX re- translation and its regulation (1). In nearly 50% of patients an- sponse in MM. Our efforts uncover a previously unidentified alyzed, mutations were documented in the following: DIS3, vulnerability in MM that should be explored clinically. a component of the exosome and predicted to increase mRNA content; FAM46C, a gene product functionally related to a reg- Author contributions: F.R., A.R., C.S., G.C.S., M.S., and J.P. designed research; F.R., W.R., ulator of protein synthesis; XBP1, an unfolded response protein and M.S. performed research; A.B., C.F., and J.A.P. contributed new reagents/analytic tools; F.R., W.R., A.B., C.F., A.R., C.S., J.A.P., G.C.S., M.S., and J.P. analyzed data; and F.R. linked to translational control; LRRK2, a eukaryotic initiation and J.P. wrote the paper. factor (eIF) 4E binding protein kinase; and, less frequently, Conflict of interest statement: C.F. is a founder and employee of Mirimus, Inc., a company eIF3B, rpL10, and rpS6KA1 (1). In addition, several signaling that has licensed shRNA technology based on the mir30 system used in this report. pathways are aberrantly activated in MM. These signaling *This Direct Submission article had a prearranged editor. pathways include the PI3K/mammalian target of rapamycin 1To whom correspondence may be addressed. Email: [email protected] or jerry. (mTOR), NF-κB, Ras, Raf, MAPK, and Janus kinase pathways, [email protected]. all of which promote proliferation, evasion of apoptosis, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. resistance to therapy (2, 3). Among these signaling pathways, the 1073/pnas.1402650111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1402650111 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 activation via the aforementioned feedback loop and show higher Because DEX is used as frontline therapy in this disease, we activity against MM cells than rapalogs (11, 14–16). Rapalogs and designed the screen to identify potential modifiers of the DEX PI3K/mTOR inhibitors have also been shown to enhance the response (Fig. 1A). Among five human MM cell lines tested for cytotoxic effects of DEX (14, 17, 18), which is an effect that can their ability to be infected by our modified pGmP lentivirus, JJN- also be obtained by sequestering eIF4E from the eIF4F complex 3, and KMS-11 were the most efficiently and reproducibly by overexpressing an inhibitory binding partner (18). Herein, we infected, with rates attaining 50–60% following one round of describe the results of a focused RNAi-based screen aimed at infection (Fig. S1A). Whereas KMS-11 cells were sensitive to identifying druggable targets among components of the trans- DEX (IC50 = 50 nM), JJN-3 cells were quite resistant to con- lation apparatus to identify DEX synthetic lethal partners in centrations as high as 3 μM(Fig. S1B), as previously reported MM. Our results define eIF4F as a target for the treatment of (19). Upon exposure to DEX, JJN-3 cells activated expression MM, identify a small-molecule inhibitor with low nanomolar of the DEX-responsive glucocorticoid-induced leucine zipper potency against human MM cells, and demonstrate that in- (GILZ) (20), indicating that DEX resistance in JJN-3 cells is hibition of translation is synthetic lethal with DEX when applied not due to defective glucocorticoid receptor (GR) signaling to MM tumor cells. (Fig. S1C). GILZ induction is mediated by DEX in our setting because it is blocked by the antagonist RU-486 (Fig. S1C). Results These results establish JJN-3 cells as a DEX-resistant cell line RNAi-Based Synthetic Lethal Screen Identifies Modifiers of DEX with a functional GR/GILZ signaling axis. Sensitivity in MM. Given the profound deregulation at the level We generated a custom, sequence-verified and arrayed, miR30- of translation that has been documented in MM (Introduction), we based shRNA library targeting amino acyl-tRNA synthetases, large chose to perform an RNAi-based screen targeting this pathway and small ribosomal proteins, initiation factors, elongation fac- to identify vulnerabilities for potential therapeutic intervention. tors, termination factors, RNA helicases, and components known Fig. 1. DEX-dependent synthetic lethal RNAi screen in the JJN-3 cell line. (A) Schematic outline of the DEX-dependent synthetic lethal screen performed in this study. A diagram of the pGmP vector and a time line of the infection and cell-harvesting schedule are presented. Puro, puromycin; SIN, self-inactivating. (B) Categories of genes evaluated in the RNAi screen. The number of genes targeted in each category is indicated in parentheses. DHX, DEAH-box. (C) Pooled synthetic lethal shRNA screen in JJN-3 cells showing changes in overall representation of 1,099 informative shRNAs during 12 d of culture.