Whole-Genome Rnai Screen Highlights Components of the Endoplasmic Reticulum/Golgi As a Source of Resistance to Immunotoxin-Media

Whole-genome RNAi screen highlights components of PNAS PLUS the endoplasmic reticulum/Golgi as a source of resistance to immunotoxin-mediated cytotoxicity

Matteo Pasettoa,1,2, Antonella Antignania,1, Pinar Ormanoglub, Eugen Buehlerb, Rajarshi Guhab, Ira Pastana,3, Scott E. Martinb, and David J. FitzGeralda,3

aLaboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264; and bDivision of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850

Contributed by Ira Pastan, February 2, 2015 (sent for review October 6, 2014; reviewed by Arthur Frankel and Wayne I. Lencer) Immunotoxins (antibody–toxin fusion proteins) target surface EF2 no longer functions at the elongation step of protein trans- antigens on cancer cells and kill these cells via toxin-mediated in- lation. Death results from a combination of events: the loss of hibition of protein synthesis. To identify genes controlling this short-lived survival proteins (e.g., Mcl1), triggering apoptosis, process, an RNAi whole-genome screen (∼22,000 genes at three and stress responses that cannot be executed because protein siRNAs per gene) was conducted via monitoring the cytotoxicity of translation is shut down (21). Regarding the constituents of the the mesothelin-directed immunotoxin SS1P. SS1P, a Pseudomonas pathway responsible for transporting the immunotoxin from the exotoxin-based immunotoxin, was chosen because it is now in surface to the cytosol, there are only a handful of experimentally clinical trials and has produced objective tumor regressions in established components. These proteins include the surface patients. High and low concentrations of SS1P were chosen to target itself, the protease furin, and KDELR2 (discussed below). allow for the identification of both mitigators and sensitizers. As In addition, within the cytosol, the diphthamide pathway re- expected, silencing known essential genes in the immunotoxin sponsible for the multistep posttranslational modification of EF2, pathway, such as mesothelin, furin, KDEL receptor 2, or members converting histidine 715 to diphthamide, is necessary (22, 23). of the diphthamide pathway, protected cells. Of greater interest Beyond these components, the total number of constituents is was the observation that many RNAi targets increased immuno- unknown, although it is under study (24). Silencing of genes toxin sensitivity, indicating that these gene products normally con- associated with the immunotoxin pathway would likely change tribute to inefficiencies in the killing pathway. Of the top sen- cell sensitivity. Furthermore, should these gene products be ame- sitizers, many genes encode proteins that locate to either the nable to regulation via small molecular drugs, alterations of killing endoplasmic reticulum (ER) or Golgi and are annotated as part of activity might be possible via chemical intervention (25, 26). the secretory system. Genes related to the ER-associated degrada- Mesothelin is expressed on mesotheliomas, as well as on other tion system were not among high-ranking mitigator or sensitizer epithelial cancers (27–32). The immunotoxin SS1P was designed candidates. However, the p97 inhibitor eeyarestatin 1 enhanced immunotoxin killing. Our results highlight potential targets for to kill cells displaying this surface protein and is currently under chemical intervention that could increase immunotoxin killing of clinical evaluation. Of note, recent results from a phase 1 trial cancer cells and enhance our understanding of toxin trafficking. combining SS1P with pemetrexed and cisplatin reported a 60%

RNAi | toxin | immunotoxin | screen | genome Significance

ntibody-based cancer therapeutics are designed to kill target To increase understanding of how antibody–toxin fusion pro- Acells, ideally causing little damage to normal cells. These teins (immunotoxins) kill cells, we used RNAi, striving to reduce molecules extend from intact IgG molecules through antibody– the expression level of all human genes. Some RNAi treatments drug conjugates to antibody–toxin fusions (1–4). When antibodies resulted in resistance to immunotoxins and some caused in- are tasked with delivering radionuclides or T cells, surface binding creased sensitivity. We focused on target genes that caused is usually sufficient. However, when delivering toxic payloads, cells to be more immunotoxin-sensitive. Results highlight antibody internalization to specific intracellular pathways plays genes that naturally protect cells from the action of immuno- a critical role in determining cellular susceptibility (5–8). toxins. Of interest, many of these genes encode proteins that Immunotoxin design includes an antibody fragment, either Fv reside in the endoplasmic reticulum and Golgi, and are part of or Fab, fused with a protein toxin (4, 9–11). The antibody frag- the cell’s secretion system. Should these gene products be ment binds a surface antigen leading to internalization followed amenable to regulation via small molecular drugs, enhance- by cleavage via the cellular protease, furin, which begins sepa- ment of killing might be possible via chemical intervention. rating the toxin from the antibody Fv (12, 13). Separation is completed by a reduction step that generates a C-terminal toxin Author contributions: A.A., I.P., S.E.M., and D.J.F. designed research; M.P., A.A., and P.O. performed research; P.O., E.B., R.G., S.E.M., and D.J.F. analyzed data; and A.A., I.P., S.E.M., fragment of 35 kDa (14). This fragment contains a KDEL-like and D.J.F. wrote the paper. sequence at the C terminus, which is necessary for cell killing Reviewers: A.F., UT Southwestern; and W.I.L., Harvard Medical School. (15). The need for a KDEL-like sequence implicates the endo- The authors declare no conflict of interest. plasmic reticulum (ER) as an essential organelle in the toxin MEDICAL SCIENCES Data deposition: The sequence reported in this paper has been deposited in the PubChem pathway and suggests that the toxin travels to the ER via a ret- database (assay ID 1117281). rograde pathway. KDEL-receptor 2 (KDELR2) has been shown 1M.P. and A.A. contributed equally to this work. to interact with Pseudomonas exotoxin (PE)-derived immuno- 2Present address: Department of Pathology and Diagnosis, Ospedale GB Rossi, 37134 toxins (16). From the ER, the C-terminal toxin fragment trans- Verona, Italy. locates to the cell cytosol, where it ADP-ribosylates elongation 3To whom correspondence may be addressed. Email: [email protected] or fitzgerd@ factor 2 (EF2). Only EF2 that is modified posttranslationally by helix.nih.gov. a multistep diphthamide pathway is susceptible to toxin-mediated This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ADP ribosylation (17–20). Cells succumb because ADP-ribosylated 1073/pnas.1501958112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1501958112 PNAS | Published online February 23, 2015 | E1135–E1142 Downloaded by guest on September 26, 2021 response rate in patients with pleural mesothelioma and a 77% Table 2. Top 24 ranked genes from the genome-wide screen for response rate at the maximum tolerated dose (33). Mesothelin is sensitizers expressed on KB cells at about 10,000 copies per cell, rendering RSA rank Symbol Gene_ID RSA rank Symbol Gene_ID these cells moderately sensitive to the SS1P immunotoxin with an IC50 of 10–20 ng/mL. 1 RPL10A 4736 13 CD48 962 RNAi results in the loss of mRNA from target genes (34). To 2 SLC33A1 9197 14 PKDREJ 10343 identify genes involved in immunotoxin-mediated killing, we 3 HDDC3 374659 15 hCG_31916 653702 undertook a whole-genome screen where three siRNAs per gene 4 RPL38 6169 16 RPL24 6152 were added before the addition of a high or low concentration of 5 BICC1 80114 17 EIF3I 8668 immunotoxin. Genes were scored as “mitigators” or “sensitizers” 6 COPB1 1315 18 RBM13 84549 based on their protection from or enhancement of immunotoxin 7 ZNF37A 7587 19 CPEB4 80315 action. To validate selected targets, additional siRNAs were 8 FUCA1 2517 20 STX19 415117 tested via dose–response analysis of immunotoxin action. Here, 9 RPLP1 6176 21 CHIT1 1118 we confirm the roles of essential genes in the immunotoxin 10 DYNLT1 6993 22 HTR4 3360 pathway and identify previously unknown members of the toxin 11 ARL1 400 23 LOC388532 388532 pathway, notably those members that usually protect cells from 12 COPE 11316 24 ARF4 378 immunotoxin action but, when reduced in expression, lead to the sensitization of cells. most significant mitigator genes determined by Haystack (Dataset Results S3). Also among the top candidates were genes that could rea- Validation of Initial RNAi Screen. To identify genes involved in sonably be expected to participate in the immunotoxin pathway. immunotoxin intoxication, a whole-genome RNAi screen was These genes were components of the endocytic pathway, in- performed on KB cells that express surface mesothelin and are cluding the clathrin coat protein AP2M1 and dynamin 2. Addi- moderately susceptible to the immunotoxin SS1P. Cells were tional members of the clathrin coat were also ranked highly transfected with three siRNAs corresponding to each of ∼22,000 (RSA: P < 0.001), including AP2A1 and AP2S1 (Dataset S1). human genes and then treated with either a “high” concentration Proapoptotic genes were also among the top candidates, in- of immunotoxin, where the silencing of essential genes would cluding a top RSA-ranked candidate, BAK1, and Haystack “ ” result in protection, or a low concentration of immunotoxin, candidate DIABLO. The identification of many expected and where silencing of inhibitory genes would result in enhanced established genes supports this approach as a reliable method to killing. ATP levels determined cell viability. Genes were ranked identify genes that modulate toxin activity. by performing redundant siRNA analysis (RSA) (35) on both mitigator and sensitizer datasets (Datasets S1 and S2). The RNAi to Identify Inhibitory Genes of Immunotoxin Action. Although top 24 candidates for each are listed in Tables 1 and 2. In bacterial toxins are highly potent, it has long been suspected that addition to selecting candidates based on the activity of their there are inefficiencies in the toxin pathway. To identify genes corresponding siRNAs, we used a computational method that responsible for diminishing immunotoxin action, we sought analyzes screen data based entirely on off-target effects (36). This candidates that resulted in increased killing. Of potential in- “ ” method, termed Haystack, was applied to both high and low terest, we found that many siRNAs sensitized cells to a low dose datasets to identify the most statistically significant genes. The top of SS1P (Fig. 1A). RSA was used to rank candidates. STRING Haystack candidates for low-dose and high-dose screens are (37) analysis indicated that the top-ranked genes (RSA: P < 0.01, reported in Datasets S3 and S4. 625 genes) were enriched significantly in known protein–protein Silencing of genes, already known to be essential for toxin interactions (P = 3.73e-8) and gene ontology (GO) terms pri- killing, should result in protection of cells if the protein products marily associated with protein synthesis and protein trafficking have a high enough turnover rate. As shown in Table 1, many (Dataset S5). Besides translation and ribosomal proteins (RPs; essential genes were ranked among the most active, including Discussion), other enriched terms were associated with ER/Golgi mesothelin (the surface target for the immunotoxin), three genes transport. In fact, of the top-ranked sensitizers in Table 2, of the diphthamide pathway (needed for the posttranslational a number of encoded proteins locate to either the Golgi or ER, modification of EF2), furin (the cellular protease responsible including COPB1, COPE, SLC33A1, and two ARF-related for toxin cleavage), and KDELR2 (a prominent ER retrieval/ B retention receptor). Notably, KDELR2 and furin were among the proteins (ARF4 and ARL1) (Fig. 1 ). ARF4 was also among the top Haystack candidates, supporting its genuine association with toxin activity. Although not as robust, additional ARF family Table 1. Top 24 ranked genes from the genome-wide screen for members exhibited activity, including ARF1 and ARF5, with mitigators multiple siRNAs enhancing the activity of low-dose toxin (Dataset S2; PubChem assay ID 1117281). RSA rank Symbol Gene_ID RSA rank Symbol Gene_ID To support the validity of top sensitizer candidates, four ad- 1 MSLN 10232 13 AP2M1 1173 ditional siRNAs to GBF1, ARF1, ARF4, and ARF5 were tested 2 KDELR2 11014 14 FURIN 5045 in the context of an immunotoxin dose–response analysis. Each 3 DNAJC24 120526 15 DPH3 285381 silenced gene resulted in sensitization of KB cells to SS1P. The 4 DPH1 1801 16 SON 6651 majority of tested siRNAs led to a greater than fourfold shift in 5 DPH2 1802 17 SSH1 54434 immunotoxin EC50 for each of these candidates and a number of 6 AFF3 3899 18 HPDL 84842 other selected genes, including SLC33A1 and COPE (Fig. S1). 7 DNM2 1785 19 LOC130355 130355 The “left” shifts in dose–response curves suggest that specific 8 GPR152 390212 20 OR51F2 119694 members of the Arf family normally control immunotoxin action 9 BAK1 578 21 DNAJC13 23317 in a negative way. Because Arf gene products are involved in 10 YAP1 10413 22 USF1 7391 coat recruitment for secretion and intracellular trafficking centered 11 FSTL5 56884 23 GCA 25801 on the Golgi and ER, they were chosen for additional consider- 12 LOC730867 730867 24 EN2 2020 ation. Further, because ARFs are reported to exhibit redundant

E1136 | www.pnas.org/cgi/doi/10.1073/pnas.1501958112 Pasetto et al. Downloaded by guest on September 26, 2021 PNAS PLUS A Sensitizer Screen Distribution 6000 5000 A 4000 3000 SCL33A1

Frequency 2000 ARF4 1000 0 -3-2-1012345

Log2 Fold Change B

Fig. 1. Overview of the genome-wide sensitizer screen. (A) Distribution of

log2-fold change values for >60,000 siRNAs (three siRNAs per gene). Top- ranked hits, including ARF4 and SLC33A1, are highlighted. (B) STRING analysis of top-ranked sensitizer candidates (RSA: P < 0.01, 635 genes) revealed enrichment for protein–protein interactions and GO terms associated with protein synthesis and Golgi vesicle formation. The depicted candidates highlight some of that connectivity and include proteins con- tributing to the enrichment for Golgi-associated GO terms.

functions (38), we conducted some experiments via the simul- taneous targeting of two specific ARF transcripts. Analysis of individual siRNAs was conducted to confirm a reduction in protein product after 48 h (Fig. S2 and Dataset S6). Those siRNAs listed in Dataset S6, leading to reduction or C complete loss of the target protein, were then used to investigate the enhancement of immunotoxin action. After a 48-h exposure to siRNAs targeting either ARF4 or ARF5, cells were challenged with SS1P and confirmed to be more sensitive to this immunotoxin than cells treated with a nontargeting siRNA (Fig. 2A). Similarly, a knockdown of ARF1 caused cells to be more sensitive to SS1P (Fig. 2B). Thus, using siRNAs and conditions that caused reductions in ARF protein levels, cells exhibited enhanced sensitivity to SS1P. To determine if these alterations were related to toxin metabolism, we treated cells with the same siRNAs but challenged them with an immunotoxin (HB21-PE40) to the transferrin receptor. Again, cells treated with siRNAs targeting ARF1, ARF4, or a combination of ARF1 + ARF4 exhibited increased immunotoxin sensitivity (Fig. 3A). Additionally, to examine pathway specificity, “ARF-silenced” cells were challenged with diphtheria toxin (DT) (Fig. S3A). DT

also kills cells via the ADP ribosylation of EF2 but escapes to the MEDICAL SCIENCES cytosol from acidic endosomes rather than from the ER. Si- lencing ARFs did not enhance DT-mediated toxicity, confirming the specificity of the Golgi/ER compartments in facilitating the Fig. 2. ARF and GBF1 knockdowns enhance SS1P cytotoxic activity. KB cells trafficking of PE. Enhancement of PE-derived immunotoxins were transfected with siRNAs targeting Arf4 (siARF4) and Arf5 (siARF5) (A), Arf1 (B), and GBF1 (siGBF1) (C) for 48 h and then treated with 10 ng/mL SS1P was consistent with an alteration in ER/Golgi trafficking, leading for 48 h. Cell viability was determined using the CellTiter-Glo assay. Values to increased toxin delivery. To address this issue directly, we are presented as a percentage relative to nontarget transfected cells treated sought to measure immunotoxin-mediated inhibition of protein with PBS. Mean values were determined for three different experiments. synthesis following ARF knockdowns. siRNAs to ARF1 + ARF4 The error bars represent the SD of the mean.

Pasetto et al. PNAS | Published online February 23, 2015 | E1137 Downloaded by guest on September 26, 2021 A notoxin binding, but more likely produced a more favorable state for toxin delivery or trafficking from the ER/Golgi to the cytosol.

Silencing of GBF1 Leading to Sensitization of Immunotoxin Action. ARF1, ARF4, and ARF5 are small GTPases that exhibit similar functionality related to coat recruitment and membrane trafficking (38). The guanine nucleotide exchange factor that acti- vates ARF1 and ARF4 is GBF1 (39–41), and cells lacking GBF1 would likely exhibit reduced ARF1 and ARF4/ARF5 activity. It was therefore of interest to note that GBF1 was also ranked among the top 1% of sensitizer genes (Dataset S1; RSA: P < 0.003). To confirm a reduction in GBF1 protein levels, RNAi was evaluated using several candidate siRNAs and Western blot analysis (Fig. S2 and Dataset S6). Subsequent experiments were conducted with the Hs-GBF1-3 siRNA (Dataset S6). Cytotox- icity assays with SS1P or HB21-PE40 confirmed that a knockdown of GBF1 resulted in enhanced immunotoxin action (Figs. 2C and 3B). Similar to the response with ARF knockdowns, cells with reduced levels of GBF1 protein were unaltered in their B response to DT (Fig. S3B). These experiments, together with the ARF knockdowns, point to a similar focus of “intrinsic resistance” to PE-based immunotoxins, which is relieved when one or more of the ARF/GBF1 proteins are reduced via RNAi. This sensitization is not seen with DT, confirming the likely participation of the Golgi/ER.

Role of ER-Associated Degradation Pathway. It has been proposed that toxins trafficking to the ER could gain access to the cytosol via the ER-associated degradation (ERAD) system (42). If ERAD were necessary, one could expect hits near the top of the mitigator list, but no hits related to the ERAD pathway were noted in top mitigators (or sensitizers). Because the participation of ERAD could not be assessed definitively from our siRNA data, we addressed the issue using the chemical inhibitor, Eeyarestatin 1 (Eer1), which interacts with p97 to regulate ERAD negatively, resulting in the accumulation of polyubiquitinated intermediates (43, 44). It has also been suggested that Eer1 inhibits Sec61-mediated protein translocation, because it targets a com- Fig. 3. ARF or GBF1 knockdowns enhance HB21-PE40 cytotoxic activity. KB ponent of the Sec61 complex that forms the membrane pore cells were transfected with siRNAs targeting Arf4, Arf5, and a combination of the ER translocon (45). KB cells incubated with Eer1 and of Arf1 (siARF1) and Arf4 (A) and GBF1 (B) for 48 h and then treated with 10 ng/mL HB21-PE40 for 48 h. The mean values were determined for three different experiments. The error bars represent the SD of the mean.

or ARF1+ ARF5 resulted in enhanced immunotoxin-mediated inhibition of protein synthesis (Fig. 4). This enhancement was consistent with an increased delivery of toxin to the cytosol. Enhancement was investigated further by tracking kinetically the amount of cell-bound and processed immunotoxin. HB21-PE40 was bound to cells at 4 °C for 30 min and then warmed to 37 °C, allowing endocytic uptake and initial intracellular processing. Samples were taken at 0, 2, and 3 h. To remove surface-bound immunotoxin and track only internalized protein, cells were washed in low pH buffer at the end of each incubation. At 2 and 3 h postwarming, there were equal amounts of intact and processed immunotoxin within cells treated with either control or Protein synthesis (% of ctr) ARF1 + ARF4 siRNAs (Fig. S4). There was no increase in the total amount of cell-associated immunotoxin or in the amount of the furin-generated 37-kDa fragment detected at 2 and 3 h in ARF1 and ARF4 double-knockdowns. This result suggested that the double-knockdown of ARF1 and ARF4 does not alter receptor levels or initial processing of internalized toxin. More- over, a similar amount of 37-kDa toxin fragment observed in the Fig. 4. 4ARF knockdowns sensitize cells to immunotoxin-mediated (IT) in- silenced and control cells suggested that enhanced delivery of hibition of protein synthesis. RNAi-transfected cells were incubated with toxin was due to a step (after furin action) that depended on 0.1 ng/mL HB21-PE40 for 24 h. Inhibition of protein synthesis was determined diminished levels of ARF1, ARF4, or ARF5. We conclude that by measuring the incorporation of 3H-leucine into cells. The values are a per- the ARF knockdowns did not alter the early events of immu- centage relative to nontarget transfected cells treated with PBS. ctr, control.

E1138 | www.pnas.org/cgi/doi/10.1073/pnas.1501958112 Pasetto et al. Downloaded by guest on September 26, 2021 immunotoxin were compared with cells treated with immuno- A no Eer1 PNAS PLUS toxin alone. Eer1 (1 μM) enhanced (approximately fivefold) the cytotoxicity of SS1P or HB21-PE40 compared with immunotoxin 100 +Eer1 alone (Fig. 5). As with previous experiments, toxin specificity was established by conducting similar experiments with DT. We found no change in DT-mediated killing using Eer1. From these data, we conclude that the p97 ATPase component of the ERAD pathway is not critical for the delivery of PE-based molecules to 50 the cytosol.

Solute Carrier/Ac-CoA Transporter. SLC33A1 is annotated as a Viability (% of ctr) transporter for Ac-CoA, and it is located in membranes of the ER (46). Functionally, SLC33A1 is linked to the unfolded pro- 0 tein response and to autophagy (46). This transporter was the 0 0.1 1 10 100 second-ranked sensitizer in the entire screen, and its role in HB21PE40 (ng/ml) causing immunotoxin resistance was supported by testing additional siRNAs in follow-up (additional siRNAs are reported in B Fig. S1). As with other RNAi treatments, we confirmed reduced no Eer1 SLC33A1 protein levels following the treatment of cells with 100 +Eer1 either Hs_SLC33A1_1 or Hs_SLC33A1_5 (Fig. S5). However, it was not obvious why reduced expression of this transporter would render cells more immunotoxin-sensitive. To confirm the result, we conducted dose–response experiments with both SS1P and HB21-PE40. Cells treated with siRNAs targeting SLC33A1 50 rendered them more sensitive to either immunotoxin (Fig. 6 A and B). When protein synthesis was measured, similar to the

ARF story, knockdowns of SLC33A1 led to enhanced im- (%Viability of ctr) munotoxin-mediated reduction in protein synthesis (Fig. 6C). However, in contrast to the ARF knockdowns, which showed no 0 alteration in response to DT, reduced levels of SLC33A1 ren- 00.1110100 dered cells resistant to DT (Fig. S6), a phenotype not seen with SS1P (ng/ml) other candidates. C Discussion no Eer1 A study published in 1978 highlighted the potency of bacterial +Eer1 toxins (47). One molecule of DT introduced into a cell can be 100 lethal, claimed the title of the paper (47). However, to achieve this result, DT was injected into cells using erythrocyte ghosts, thereby bypassing endocytosis. Here, we report on the silencing of genes that render cells more sensitive to PE-based immunotoxin killing. That these experiments were successful confirms 50 inefficiencies in the immunotoxin pathway. We conclude that some gene products “normally” diminish toxin action, either directly or indirectly. Thus, these gene products could be con- Viability (% of ctr) sidered “protective” of the cell, at least with regard to PE-based 0 immunotoxins. It is noteworthy that several PE immunotoxins 00.010.11 10 are currently being evaluated as cancer therapeutics in clinical trials. Most trials have administered immunotoxins as single DT (ng/ml) agents. However, with the design of more advanced trials, it is anticipated that combination therapy will be used. Thus, by Fig. 5. Eer1 enhances immunotoxin activity. KB cells were incubated for μ identifying gene products that are responsible for treatment re- 24 h with HB21-PE40 in the presence or absence of 1 M Eer1 (A), with SS1P sistance, it may be possible to choose compounds that target the with or without Eer1 (B), or with DT plus or minus Eer1 (C). Cell viability was determined using the CellTiter-Glo assay to measure ATP levels. specific gene products identified in our screen, thereby enhancing therapeutic outcomes. Of our top sensitizing candidates, a number locate to the ER toxicity associated with BFA. Here, we report an opposite phe- and Golgi, suggesting that these organelles facilitate toxin me- notype. Arf4, Arf1, and GBF1 apparently direct PE-based im- tabolism at less than full efficiency. Further, when RNAi-treated munotoxins into a pathway of reduced efficiency. When any of (e.g., targeting ARF1/ARF4/GBF1) cells were challenged with these genes was silenced, cells were more sensitive to immuno- DT, there was no change in sensitivity to this toxin. Thus, many

toxin killing. MEDICAL SCIENCES of our hits appear particular to the pathway taken by PE-based A study of ricin and PE toxicity sought gene products that, immunotoxins. Paradoxically, the ER/Golgi is essential for toxin transport (note that KDELR2 is essential for killing and is very when silenced, rescued cells from toxin-mediated inhibition of high on the mitigator list), and yet some of its constituents protein synthesis (24). Hits from this assay would be equivalent contribute to inefficiencies. to our mitigator list with two distinctions: Moreau et al. assayed Recent experiments exploring the mechanism of Brefeldin for rescue of protein synthesis inhibition at 8 h, whereas we A (BFA) toxicity implicated Arf4 as an active participant in that assayed for protection in a 72-h viability assay, and they used pathway (41). Cells with Arf4 knockdowns (but not other Arfs) native PE, whereas we used PE-based immunotoxins (24). were BFA-resistant, suggesting that Arf4 was mediating the Comparing our list with the list of Moreau et al. revealed little

Pasetto et al. PNAS | Published online February 23, 2015 | E1139 Downloaded by guest on September 26, 2021 overlap [∼7% pairwise overlap between the 178 “validated” hits in the Moreau study and the top mitigators presented here A (RSA: P < 0.01)]. Limited concurrence between the studies is not surprising, given the differences in assay systems and the notorious rate of false- positive results in RNAi screens (48, 49). Nonetheless, a few key genes overlapped, including KDELR2 and CUL3, which was found to be important for endosome-to- Golgi retrograde trafficking and PE activity. Other related screens include a recent one by Gilbert et al. (50), where cells that had been subjected to CRISPR-mediated gene manipula- tion were challenged with a cholera–DT fusion. Comparing hits of their top 500 mitigators and sensitizers with ours showed concurrence of ∼30 genes. Notable common sensitizers in- cluded COPs and RPs, as well as selected hits, including high ranked mitigators (KDELR2) and sensitizers (COPB1, GBF1, EXOSC6, and EXO1). Toxins that traffic through the ER are potential substrates for ERAD (42). Toxins, mimicking unfolded proteins, would be transported to the cytosol as part of the cell’s quality control system. Evidence that ricin, shiga-like toxin, cytolethal distending toxins, and cholera toxin interact with the ERAD pathway al- B ready exists (51–54). A review of the mitigator list found no clear evidence that this pathway was essential for PE-based immunotoxins. Thus, we sought to address the issue using a chemical inhibitor of ERAD. Eer1 interacts with p97 to regulate ERAD negatively, resulting in the accumulation of polyubiquitinated intermediates (43, 44). Our results, using KB cells as a model system, failed to confirm an essential role for p97, although our studies were limited in scope and now await an expanded in- vestigation of PE–toxin interactions with the ERAD pathway. Further, we note that Aletrari et al. (55) reported that Eer1 had no role as an inhibitor of Shiga toxin dislocation from the ER; rather, they suggested that Eer1 protected cells by disrupting anterograde trafficking. In our experiments, we noted enhanced immunotoxin action in the presence of Eer1 (Fig. 5). We also noted enhancement when components of the secretory appara- tus of the ER/Golgi were down-regulated via RNAi. Additional experiments will be needed to determine if the Eer1 and RNAi enhancements are focused on a common cellular component of the secretion pathway. GO analysis identified a significant enrichment for translation- associated processes in the enhancer screen (Dataset S5). For C example, ∼10% of the top 200 sensitizers were annotated as genes encoding RPs (Dataset S7 lists the top 19 RP hits). None were found as top candidates in the mitigator screen. Although little is known about the roles played by these RPs in the genesis and maintenance of cancer, the few existing reports indicate higher expression levels of certain RPs in malignant cells. In addition to structural roles, some RPs clearly have other functions in cellular metabolism, including interactions with MDM2 (or its human counterpart), promoting downstream interactions with p53 (56). Overexpression of RPL24, RPL39, or RPS28 has each been reported in breast cancer (57–59). High levels of RPL36 were noted in liver cancer (60), high levels of RPS16 were noted in androgen-independent prostate cancer (61), and high levels of RPS23 were noted in colorectal cancer (62). Overall, our data support the assertion that several specific ARFs, RPs, and SLC33A1 normally protect cells from the action of PE-based immunotoxins. To inquire if these gene products protect cells from other stresses, we searched for reports de- scribing loss or reduced levels of these same proteins. Loss of Fig. 6. SLC33A1 knockdowns result in enhanced SS1P activity. KB cells were RPs renders individuals susceptible to Diamond–Blackfan ane- transfected with a nontarget siRNA or one of two siRNAs targeting SLC33A1 mia and myelodysplastic syndrome (63). Further, reduced levels (siSLC33A1-1, siSLC33A1-5) for 48 h and then treated with 10 ng/mL SS1P (A) or 1 ng/mL HB21-PE40 (B) for 48 h. Mean values were determined for three of SLC33A1 result in pathogen-driven inflammation and cancer, different experiments. The error bars represent the SD of the mean. (C) leading to premature death of affected mice (64). Thus, we can KB3-1 cells were transfected with a nontarget siRNA or the siRNA SLC33A1-1 conclude that the RPs and SLC33A1 may be generally protective for 48 h and then treated with 10 ng/mL SS1P for 24 h. Inhibition of protein of cells. The case for ARFs appears more situation-specific. The synthesis was determined as described. challenge lies in how to target these gene products clinically.

E1140 | www.pnas.org/cgi/doi/10.1073/pnas.1501958112 Pasetto et al. Downloaded by guest on September 26, 2021 Although amino acid treatments may improve anemias, immu- Technologies) RNAiMax (0.05 μL) was added to wells containing siRNA (0.8 PNAS PLUS notoxin therapy will require drugs that destabilize RPs (65). To pmol). Lipid and siRNA were allowed to complex for 45 min before addition probe the role of ARFs, new chemical inhibitors are needed (66). of 500 cells in DMEM and 20% FBS to yield final transfection mixtures Existing compounds apparently lack both potency and specificity. containing 20 nM siRNA in DMEM and 10% FBS. The screen was conducted using the Ambion (Life Technologies) (70) Silencer Select Version 4 genome- As immunotoxins progress through clinical development, it is wide collection. This library targets >21,000 human genes with three siRNAs likely that patients will benefit from combinations that include per gene. Toxin (“high dose,” ∼EC90, 13 ng/mL; “low dose,” ∼EC30, 3 ng/mL; agents that enhance immunotoxin action (33). Some of these or “no dose,” 0 ng/mL) was added to the plate 48 h posttransfection, and agents may overcome delivery barriers, promoting improved viability (CellTiter-Glo; Promega) was assayed 72 h later. Ambion Silencer access of immunotoxins to tumor cells, and some may blunt Select Negative Control No. 2 (Life Technologies) was incorporated on all immune responses and thereby allow additional cycles of ther- screening plates. Qiagen’s AllStars Cell Death control was a positive trans- apy. Here, we suggest a previously unidentified set of targets. fection control on low-dose and 0-ng/mL plates. Ambion Silencer Select These proteins, located in the ER/Golgi, apparently normally MSLN siRNA (catalog no. s11968; Life Technologies) was used as a positive reduce the activity of PE-based immunotoxins. Those targets control for high-dose plates. highlighted in our study, including ARF1, ARF4, ARF5, and To select candidates whose knockdown mitigated toxin activity, RSA (35) GBF1, point to the secretory arm of the Golgi/ER as a potential was performed on the high-dose dataset after normalization of each siRNA to the median negative control value. To select a candidate whose knock- complex for targeting via chemical intervention. down enhanced toxin activity, the log2 ratio of no-dose cell viability [% nontarget oligo (siNeg)] to low-dose toxin-treated cell viability (% siNeg) Materials and Methods was calculated for each siRNA. RSA was then performed on the ratios to rank Cell Culture. KB cells (67) were cultured in DMEM supplemented with gene candidates in terms of their ability to sensitize KB cells to low-dose 10% (vol/vol) FBS. immunotoxin. RSA rankings can be found in Datasets S1 (mitigators) and S2 (sensitizers). Screening data can be found on the PubChem database (assay Reagents and Antibodies. Two immunotoxins, SS1P and HB21-PE40, were ID 1117281). Haystack analysis was performed on low-dose and high-dose purified in our laboratory (68). Anti-ARF4 was from Proteintech, anti-ARF1 datasets as described (36). (1D9) was from Abcam, anti-GBF1 was from BD Transduction Laboratories, and anti-SLC33A1 was from Abnova. The anti-PE monoclonal M40-1 was de- Validation RNAi Experiments. To knock down mRNAs in KB cells, 5,000 cells scribed previously (69). Eer1 was purchased from Santa Cruz Biotechnology. were transfected with HiPerFect HTS Reagent (Qiagen). To silence the target, the following final concentration amounts of siRNA (Dataset S6) were used: Immunotoxin Cleavage and Western Blot. KB cells were transfected with siRNA 15 nmol/L (to silence ARF4, ARF5, and SLC33A1) and 30 nmol/L (to silence for 48 h. At 48 h, HB21-PE40 (1 μg/mL) was added to cells and incubated on ARF1 and GBF1). The final volume per well for 96-well experiments was 150 μL. ice for 30 min. Cells were washed with PBS, changed to fresh media, and After 48 h of transfection, the cells were treated with immunotoxin or other incubated at 37 °C for the indicated times. Cells were treated with trypsin at toxic agents as indicated. All siRNA experiments used a nontargeting siRNA 4 °C and washed in a low-pH glycine buffer to remove surface immunotoxin. (Qiagen) as a negative control. Cell viability was measured using a CellTiter-Glo Cells were solubilized in radioimmunoprecipitation assay buffer with pro- luminescent cell viability assay (Promega), whereas protein synthesis was quan- – 3 tease and phosphatase inhibitors. Proteins were loaded onto NuPAGE 4 12% tified by incubating cells with 2 μCi/mL H-leucine (PerkinElmer). Bis-Tris gels (Life Technologies) and transferred to nitrocellulose membranes. ACKNOWLEDGMENTS. This work was supported by the Intramural Research Whole-Genome RNAi Screen. Transfections were performed in 384-well plates. Program of the NIH, National Cancer Institute, Center for Cancer Research, For transfections, 20 μL of serum-free media containing Lipofectamine (Life and via the NIH Director’s Challenge Innovation Award.

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