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 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 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. 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 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 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 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 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 , initiation factors, elongation fac- to identify vulnerabilities for potential therapeutic intervention. tors, termination factors, RNA , 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. Depletion of shRNAs was calculated as the relative abundance in vehicle (DMSO)-treated cells divided by the relative abundance in 100 nM DEX-treated cells. Values are plotted as the average of triplicate values for each representation. The locations of the shRNAs against MCL1, firefly luciferase (FLuc), and the scrambled (Scr) controls are indicated. The horizontal dotted line represents three SDs from the population mean. (D) Pie chart illustrating the number and validation outcome of shRNAs identified in the DEX-dependent RNAi screen.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1402650111 Robert et al. Downloaded by guest on September 27, 2021 to regulate protein synthesis (Fig. 1B and Dataset S1). The library not eIF4A2 or eIF4G3, as potential modifiers of DEX sensitivity consisted of 1,534 unique shRNAs targeting 268 genes (including in JJN-3 cells. controls; SI Text 1 and Fig. S2) cloned into pGmP (Fig. 1A and Dataset S1). The library was transduced into JJN-3 cells and Pharmacological Targeting of eIF4F Sensitizes MM Cells to DEX. We cells cultured for 12 d in the presence of vehicle or DEX. sought to determine if we could phenocopy the sensitization of Changes in shRNA representation were determined by deep JJN-3 cells to DEX obtained by RNAi-mediated suppression of sequencing of shRNA sense strands amplified from genomic eIF4F using small-molecule inhibitors. Efforts toward “drug- DNA at day 0 [time (T) 0] and day 12 (T12). Each experimental ging” the eIF4F complex have identified a number of compounds time point was performed on three independent biological rep- capable of blocking eIF4E–cap interaction, interdicting eIF4E–eIF4G licates, and correlation coefficients between replicates ranged association, or inhibiting eIF4A-mediated helicase activity (4) from 0.6 to 0.96 (Dataset S1 and Table S1). (Fig. 3A). JJN-3 cells are exquisitely sensitive to the eIF4A < B In total, 1,099 shRNAs (72% of the library) were detected helicase inhibitor silvestrol, displaying an IC50 10 nM (Fig. 3 A above background in all three biological replicates at T0. At T12, and Fig. S4 ). In comparison, the IC50 of a second unrelated we identified 18 potential DEX-dependent synthetic lethal can- eIF4A inhibitor, hippuristanol (Hipp), was ∼100 nM (Fig. 3B). – ∼ μ didates for which the representation of two independent The eIF4E eIF4G interaction inhibitors, 4E1RCat (IC50 of 6 M) ∼ μ shRNAs was reduced in the presence of DEX (Fig. 1 C and D and 4EGI-1 (IC50 of 20 M), were the least active of the and Dataset S2). Among these candidates, seven encoded RNA compounds tested (Fig. 3B). For comparison, we assessed JJN-3 helicases, 10 coded for core components of the translation ap- sensitivity to the mTORC KI, OSI-027, and the dual-specificity paratus, and one encoded a translational regulator (GCN1L1) PI3K/mTOR KI, NVP-BEZ235, and found that JJN-3 cells were ∼ (Fig. 1D). During the analysis process, we recognized that sup- more sensitive to NVP-BEZ235 (IC50 of 20 nM, compared with ∼ pression of all three eIF4F subunits synergized with DEX and the IC50 for OSI-027 of 500 nM), none of which displayed the focused our validation efforts on the eIF4F cap-binding protein potency obtained with silvestrol (Fig. S4B). complex (Fig. 1D). A series of extended titrations combining eIF4F inhibitors with DEX revealed significant synergy (combination index value eIF4F Is a Genetic Modifier of the DEX Response in JJN-3 Cells. In- lower than 0.25) for the DEX/silvestrol combination at con- dividual testing of shRNAs targeting the three eIF4F subunits centrations of silvestrol as low as 0.4 nM and over a 2.5-log10 GENETICS confirmed their synthetic lethal relationship with DEX (Fig. 2A). concentration range (Fig. 3C). NVP-BEZ235 also synergized We compared the extent of shRNA depletion in the presence of with DEX, but not as profoundly as silvestrol over the con- DEX with the knockdown efficiency of the shRNAs toward their centrations tested (Fig. 3C). Strong synergy was also observed targets and found that the more potent the shRNA, the greater between Hipp and DEX at concentrations of Hipp ≥25 nM, was the sensitization to DEX (Fig. 2 A–C). There are two cel- moderate synergy was noted with OSI-027 and DEX over a wide lular isoforms of eIF4A and eIF4G that can be incorporated into concentration range, and 4E1RCat and 4EGI-1 synergized with the eIF4F complex with some potential nonoverlapping roles in DEX at concentrations between 50 and ∼750 nM (Fig. S5). These vivo (21–23), yet only shRNAs to eIF4A1 and eIF4G1 were results were not a peculiarity of JJN-3 cells, because we re- identified in the primary screen as being synthetic lethal with produced our findings in a different MM cell line, KMS-11 (SI DEX (Dataset S2). Testing of shRNAs to eIF4A2 and eIF4G3 Text 2 and Fig. S6). Taken together, these results indicate that indicated that suppression of eIF4A2 or eIF4G3 is tolerated in silvestrol, an inhibitor of eIF4F helicase activity, shows nano- JJN-3 cells and is not synthetic lethal with DEX (Fig. S3). Taken molar potency as a single agent and synergizes with DEX in together, our data implicate eIF4E, eIF4A1, and eIF4G1, but MM cells.

Fig. 2. Suppression of eIF4F subunits sensitizes JJN-3 cells to DEX-induced cell death. (A) Changes in the representation of JJN-3 cells infected with the indicated pGmP-shRNA constructs in the presence of vehicle (DMSO, Upper) or 100 nM DEX (Lower) over the course of 12 d (n = 3). Error bars represent ± SEM. (B) Immunoblots of whole-cell lysates of puromycin-selected JJN-3 cells infected with the indicated shRNAs. (C) Box plot comparing eIF4F subunit knockdown efficiency with DEX sensitization in JJN-3 cells.

Robert et al. PNAS Early Edition | 3of6 Downloaded by guest on September 27, 2021 in eIF4F activity (4, 24). Accordingly, silvestrol has been docu- mented to preferentially inhibit the translation of mRNAs with elevated 5′ UTR secondary structure (25, 26). We therefore investigated the silvestrol response of several known eIF4F- responsive transcripts [MYC, myeloid cell leukemia 1 (MCL1), BCL2, and BCLXL] encoding products key to tumor cell survival (16, 27, 28). A significant reduction in MCL1 and MYC protein levels was observed in JJN-3 cells exposed to a short-term pulse (5 h) of silvestrol, Hipp, and 4E1RCat (Fig. 4A). This response correlated with a reduced association of MCL1 and MYC mRNA with polysomes in silvestrol-treated cells (Fig. 4B), with little change in mRNA levels (Fig. S8A). The DEX/silvestrol combination did not reduce MCL1 protein levels further beyond those observed with silvestrol alone (Fig. S8B, compare lanes 3 and 4), indicating that DEX, per se, is not influencing MCL1 levels. Cell death was not observed in JJN-3 cells within the experimental time period and at the concentrations of silvestrol used (Fig. S8C). In contrast, BCLXL and BCL2 protein levels changed modestly at the highest tested concentrations of silves- trol and Hipp (Fig. S8D). MCL1 is essential for survival of MM cells, and its overexpression is associated with relapse and a shortened survival period (29). Indeed, elevating MCL1 levels in the DEX-responsive KMS-11 cell line can increase resistance to DEX (Fig. S9). Ectopic over- expression of MCL1 (Fig. 4C) also altered the sensitivity of MM cells to silvestrol by increasing the IC50 twofold (Fig. 4D)and blunted the DEX/silvestrol synergy (Fig. 4E). Because IL-6 can induce expression of MCL1 in MM and favor MM cell survival Fig. 3. Pharmacological inhibition of eIF4F synergizes with DEX in JJN-3 (30) in some cases, we tested whether IL-6–induced MCL1 levels cells. (A) Schematic diagram illustrating the sites of eIF4F inhibition by – compounds used in this study. 4EGI-1 and 4E1RCat inhibit eIF4E–eIF4G in- could still be blunted by silvestrol. Exposure of the IL-6 responsive teraction, Hipp prevents eIF4A from binding RNA, and silvestrol leads to MM.1S cell line to paracrine IL-6 increased MCL1 levels, and this depletion of eIF4A from the eIF4F complex (4). (B) Pharmacological in- response was still inhibited by silvestrol (Fig. S10). We also assessed hibition of eIF4F in MM. Cells were exposed to increasing concentrations of if suppression of MYC could synergize with DEX in JJN-3 cells. compound for 48 h, and viability was determined (n = 3). Error bars repre- Inhibition of MYC production using the BET(bromo and extra sent ± SEM. (C) DEX-dependent synergy with silvestrol (Upper) or NVP- terminal)-bromodomain inhibitor JQ1 (Fig. 5 A and B) was asso- BEZ235 (Lower) in JJN-3 cells. CI, combination index. (D) Primary human MM ciated with significant synergy with DEX (Fig. 5C). Taken together, cells are sensitive to silvestrol (Sil). Primary tumor cells were exposed ex vivo these results identify MCL1 and MYC as eIF4F-responsive targets to the indicated concentrations of silvestrol for 24 or 48 h, after which point cell viability was determined by flow cytometry as indicated in Experimental whose levels can influence sensitivity to DEX. + Procedures. Presented is the percentage of the CD138 cell population sur- Discussion viving following drug exposure relative to DMSO controls. (E) Cells from patients whose tumor was refractory to DEX are resensitized by silvestrol ex Translation, as well as several upstream regulatory nodes, is of- vivo. Primary tumors cells were exposed ex vivo to the indicated concen- ten usurped in MM (Introduction). Herein, we report on a fo- trations of DEX (nanomolar), silvestrol (nanomolar), or a combination of cused shRNA screen that identified several vulnerabilities that both for 48 h, at which point cell viability was determined by flow cytom- could be therapeutically explored in this disease (Fig. 1). It is etry. Presented is the percentage of the cell population surviving following striking that among the targets identified as being synthetic lethal = ± drug exposure relative to DMSO controls (n 3). Error bars represent SEM. with DEX, suppression of all three subunits of the eIF4F com- plex was among the “hits” (Fig. 1). We independently validated We also assessed silvestrol’s activity against primary patient- these results by pharmacological targeting of eIF4F in MM in the presence of DEX. One difference we observed between the derived myeloma samples and observed significant depletion of + shRNA-based validation and experiments involving small-mole- CD138 plasma cells following 48 h of exposure to 50 nM silvestrol cule inhibitors was that long-term (12 d) RNAi-mediated sup- ex vivo (Fig. 3D). We also assessed what effect silvestrol and DEX pression of eIF4F subunits in JJN-3 cells was not as toxic as would have against primary tumor cells that no longer responded to small-molecule–mediated inhibition of eIF4F on a shorter time DEX (Fig. 3E). Our results indicate that silvestrol as a single agent + scale (∼48 h). Although we cannot formally rule out potential selectively affects CD138 cell viability and can resensitize + off-target drug effects contributing to drug toxicity, our finding DEX-resistant CD138 cells to DEX ex vivo. These results were may also be a consequence of incomplete suppression of eIF4F not due to a general cytotoxic property of silvestrol, because the − subunits by the shRNAs used. It is interesting that we observed CD138 cell population remained unaltered upon drug exposure synergy between eIF4A1 or eIF4G1 suppression and DEX, but (Fig. 3E). Three nontransformed human cell lines, as well as not when eIF4A2 or eIF4G3 was suppressed (Fig. 1 and Fig. S3). normal peripheral blood mononuclear cells, were found to be Although we cannot rule out insufficient knockdown of eIF4A2 relatively resistant to silvestrol and DEX, with little to no DEX/ and eIF4G3 levels as being responsible for this lack of response, silvestrol synergy noted (Fig. S7). Taken together, these results the results are consistent with previous data alluding to functional identify silvestrol as a potent single agent against MM cells and differences between the eIF4A and eIF4G isoforms (21–23). as being capable of resensitizing MM cells to DEX. Previous studies have characterized an addiction of MM cells to PI3K/mTOR signaling (3) and MYC (16), both of which Targeting eIF4F Acutely Curtails Production of Myeloid Cell Leukemia 1 are pathways intimately linked to translational control. A and MYC in MM. The translation of mRNAs with elevated sec- previous report documented the ability of 50 μM 4EGI-1 to trigger ondary structure within their 5′ UTRs is sensitive to alterations apoptosis in several MM cells as a single agent and demonstrated

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1402650111 Robert et al. Downloaded by guest on September 27, 2021 Fig. 4. Silvestrol inhibits production of MCL1 and MYC in MM cells. (A) Western blot monitoring MCL1 and MYC levels following acute treatment of JJN-3 cells with eIF4F inhibitors. Cells were incubated with the indicated concentrations of silvestrol and Hipp, 4E1RCat (25 μM), or 4EGI-1 (50 μM) for 5 h, at which point extracts were prepared, fractionated on SDS/PAGE, and transferred to PVDF membranes. Immunoblots were probed with antibodies to the indicated proteins. (B) Polysome profile analysis of JJN-3 cells exposed to vehicle or 20 nM silvestrol for 1 h. Shown below the polysomes are the RT-quantitative PCR results demonstrating the distribution of MCL1 and MYC mRNA throughout the polysome gradients. (C) Expression levels of MCL1 in JJN-3 cells infected with lentiviral (LEGO) or LEGO-MCL1 lentivirus. Following infection, cells were sorted by FACS. Forty-eight hours later, the infected population was lysed, fractionated by SDS/PAGE, and transferred to PVDF membranes. Western blots were probed with antibodies to the indicated proteins. (D) Change in representation of JJN-3 cells infected with LEGO or LEGO-MCL1 and grown in the presence of the indicated concentrations of silvestrol (n = 3). Error bars

represent ± SEM. (E) Ectopic expression of MCL1 blunts DEX/silvestrol synergy. GENETICS

a reduction in MCL1 and BCLXL, but not BCL2, levels (31). We (37). The ability of silvestrol to affect several biological processes also find that 4EGI-1 is effective against MM cells; however, simultaneously is a key distinguishing feature of inhibiting among the compounds tested, it was the least potent, displaying eIF4F activity. In sum, our results demonstrate that targeting an IC50 of 20 μM against JJN-3 cells (Fig. 3B). In contrast, blocking eIF4F helicase activity was a more effective means of sensitizing JJN-3 cells to DEX. An advantage of using silvestrol over mTOR or PI3K/TOR KIs is that elevated eIF4E levels can lead to PI3K/TOR KI resistance (32), and by inhibiting eIF4A, one targets downstream of this resistance node. We found that silvestrol was quite potent as a single agent against all MM cells and primary MM samples tested (Fig. 3 and Fig. S6 C and D). Silvestrol was also capable of increasing the sensitivity of JJN-3 and KMS-11 cells to DEX. Although the molecular basis of DEX resistance in the clinic is not completely understood, several studies indicate that a source of acquired resistance in leukemic cells is due to low or defective GR (33–36). In addition, long-term DEX treatment can lead to acquired re- sistance through mechanisms that may involve epigenetic events. Although we have not measured the absolute number of GRs in JJN-3 cells, the GR/GILZ signaling axis appears intact and functional (Fig. S1C). In a clinical setting of DEX resistance, silvestrol resensitized MM cells to DEX ex vivo (Fig. 3E), al- though we do not have a molecular understanding of the basis of DEX resistance in this case. The clinical challenge will be to determine the optimal setting in which to use silvestrol (or a re- lated compound) to take advantage of its single-agent potency and ability to augment DEX effectiveness. Although large-scale characterization of changes in the trans- latome would be required to identify all silvestrol-responsive tran- scripts, our studies identify MCL1 and MYC as being important to silvestrol’s mechanism of action. Nonetheless, we cannot rule out the contribution from other silvestrol-responsive transcripts. The fact that silvestrol inhibits MCL1 and MYC production and that individual suppression of MCL1 or MYC expression Fig. 5. Suppression of MYC synergizes with DEX in JJN-3 cells. (A)JQ1 affects MYC production in JJN-3 cells. JJN-3 cells were exposed to vehicle or synergizes with DEX (Fig. 5 and Figs. S2A and S6)indicate the indicated concentrations of JQ1 for 16 h, lysed, prepared for Western that silvestrol is exerting its sensitization effect through al- blotting, and probed with the indicated antibodies. (B) Response of JJN-3 tering levels of these (and possibly other) translational targets. cells to JQ1. Cells were incubated in the presence of JQ1 for 48 h, and via- We note that MCL1 has also been implicated as a modifier of bility was assessed (n = 3). Error bars represent ± SEM. (C) Synergy between glucocorticoid-induced cell death in acute lymphoblastic leukemia DEX and JQ1.

Robert et al. PNAS Early Edition | 5of6 Downloaded by guest on September 27, 2021 the eIF4F/eIF4A translational node is an effective approach to V-phycoerythrin (BD Pharmingen) and were analyzed for apoptosis by flow curtail survival of MM cells, silvestrol is a potent single agent cytometry (FACSCalibur; Becton Dickinson). against MM, and silvestrol (or related compounds) could prove to be an attractive adjunct to DEX therapy. shRNA Library Design and Synthetic Lethal RNAi Screen. Information regarding the construction of the human shRNA library targeting the translation ap- Experimental Procedures paratus, establishment of parameters for the synthetic lethal RNAi screen, and Cell Lines and Primary Myeloma Samples. JJN-3, KMS-11, RPMI8226, U266B1, analysis of deep sequencing data is provided in SI Experimental Procedures. INA-6, MM.1S, MM.1R, and OPM1 cell lines were maintained in RPMI sup- plemented with 10% (vol/vol) FBS, penicillin/streptomycin, and glutamine. BJ, Western Blots. Western blots were performed as previously described (26). IMR90, and W138 cell lines were grown in DMEM supplemented with 10% Details are provided in SI Experimental Procedures. (vol/vol) FBS, penicillin/streptomycin, and glutamine. Cells were routinely split 1:3 every 2–3 d and discarded after >3 wk in culture. The 293T/17 cells were In Vitro Fitness Assay and Median Effect Analysis. Median effect analysis maintained in DMEM supplemented with 10% (vol/vol) FBS, penicillin/ was performed essentially as described (26). Details are provided in SI streptomycin, and glutamine. Experimental Procedures. For apoptosis assays of primary patient samples, bone marrow samples from patients with MM were harvested following a McGill University Health Centre Institutional Review Board-approved informed consent protocol, and ACKNOWLEDGMENTS. We thank Dr. Sidong Huang for critical reading of the manuscript. This work is supported by grants from The Quebec mononuclear cells were plated in Iscove’s modified Dulbecco’s medium Consortium for Drug Discovery (to G.C.S. and J.P.), the Richard and Edith supplemented with 15% (vol/vol) FCS in the presence of vehicle alone or the Strauss Foundation of Canada (to M.S.), the National Institutes of Health indicated concentrations of DEX and/or silvestrol. Following 24–48 h of in- (Grant GM-073855 to J.A.P.), and the Canadian Institutes of Health Research cubation, cells were double-stained with anti–CD138-Cy5 and Annexin (Grant MOP-106530 to J.P. and Grant MOP-123503 to M.S.).

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