Evolutionarily conserved ERH controls CENP-E PNAS PLUS mRNA splicing and is required for the survival of KRAS mutant cancer cells

Meng-Tzu Wenga,b,c, Jih-Hsiang Leea, Shu-Chen Weid, Qiuning Lia, Sina Shahamatdara, Dennis Hsua, Aaron J. Schettere, Stephen Swatkoskif, Poonam Mannang, Susan Garfieldg, Marjan Gucekf, Marianne K. H. Kima, Christina M. Annunziataa, Chad J. Creightonh, Michael J. Emanuelei, Curtis C. Harrise, Jin-Chuan Sheud, Giuseppe Giacconea, and Ji Luoa,1

aMedical Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; bGraduate Institute of Clinical Medicine, National Taiwan University, Taipei 100, Taiwan; cFar-Eastern Memorial Hospital, Taipei 220, Taiwan; dDepartment of Internal Medicine, National Taiwan University Hospital and College of Medicine, Taipei 100, Taiwan; eLaboratory of Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; fProteomics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; gConfocal Microscopy Core Facility, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; hDepartment of Medicine and Dan L. Duncan Cancer Center Division of Biostatistics, Baylor College of Medicine, Houston, TX 77030; and iDepartment of , Harvard Medical School and Brigham and Women’s Hospital, Boston, MA 02115

Edited by Bert Vogelstein, Johns Hopkins University, , MD, and approved November 12, 2012 (received for review June 1, 2012) Cancers with Ras mutations represent a major therapeutic prob- anaphase-promoting complex (APC/C) that coordinately maintain lem. Recent RNAi screens have uncovered multiple nononcogene the fidelity of segregation (6). Symmetrical distribu- addiction pathways that are necessary for the survival of Ras mu- tion of during is critical for genomic stability tant cells. Here, we identify the evolutionarily conserved en- and cell survival (8, 9). During metaphase, chromosomes con- hancer of rudimentary homolog (ERH), in which depletion causes gression from spindle poles to the metaphase midplate is driven by greater toxicity in cancer cells with mutations in the small GTPase the plus end-directed protein E (CENP-E) (10). KRAS compared with KRAS WT cells. ERH interacts with the spliceo- Unattached activate spindle assembly checkpoint

some protein SNRPD3 and is required for the mRNA splicing of the such as budding uninhibited by benzimidazoles 1 homolog CELL BIOLOGY mitotic CENP-E. Loss of ERH leads to loss of CENP-E (Bub1), MAD3/BUB1-related protein kinase (BubR1), and mitotic and consequently, chromosome congression defects. Gene expres- arrest deficient 2-like protein 1 (MAD2), which in turn, inhibit the sion profiling indicates that ERH is required for the expression of activity of APC/C to delay anaphase onset until all sister multiple , and the signature result- are bioriented and properly attached to opposite spindle poles (11). ing from ERH down-regulation inversely correlates with KRAS sig- Many mitotic proteins are degraded by APC/C on mitotic exit. natures. Clinically, tumor ERH expression is inversely associated CENP-E is one such protein, and it is degraded on mitosis exit and with survival of colorectal cancer patients whose tumors harbor resynthesized in the next S-phase (12). Thus, the proper expression KRAS mutations. Together, these findings identify a role of ERH in and turnover of CENP-E during each cell cycle is necessary for mRNA splicing and mitosis, and they provide evidence that KRAS chromosome congression and genomic stability (13, 14). mutant cancer cells are dependent on ERH for their survival. In this report, we identify a candidate Ras synthetic lethal gene, enhancer of rudimentary homolog (ERH). ERH is a highly synthetic lethality | spliceosome conserved gene originally identified in (15), and it has been implicated to play a role in nuclear gene expression (16– he Ras family of small GTPases is mutated in a significant 18). Here, we show that ERH interacts with the Sm protein Tfraction of human cancers, with high frequencies of muta- SNRPD3, and it plays a critical role in the mRNA splicing and tions in v-Ki-ras2 Kirsten rat sarcoma viral homolog therefore, expression of CENP-E. KRAS mutant colorectal (KRAS) found in colon, lung, and pancreatic cancers (1–4). Ras cancer (CRC) cells are more sensitive to the depletion of ERH proteins are activated by growth factor receptors, and they, in turn, protein. Consistent with this finding, low ERH expression is as- activate a number of downstream effector pathways to coordinate sociated with better survival in cancer patients whose tumors cell proliferation, survival, and migration. Somatic mutations in harbor KRAS mutations. Our findings suggest that targeted in- Ras frequently lead to its constitutive activation, which in turn, activation of splicing machinery could be exploited to thera- drives malignant growth. Cancer cells harboring mutations in peutically restrict the malignancy of Ras-driven cancer. KRAS often exhibit the classic behavior of oncogene addiction: they become dependent on the KRAS oncogene for growth and Results survival and therefore, are hypersensitive to the loss of KRAS Ras Mutant Cells Are Hypersensitive to ERH Depletion. We identified protein (5). Efforts to pharmacologically inactivate mutant KRAS ERH as a candidate KRAS synthetic lethal gene from a - have been unsuccessful thus far. To identify additional genetic de- pendencies in Ras mutant cells, we previously conducted a genome- wide shRNA synthetic lethal screen in isogenic KRAS mutant and WTcells(6).Inthisscreen,weidentified a surprisingly diverse set of Author contributions: M.-T.W. and J.L. designed research; M.-T.W., J.-H.L., S.-C.W., Q.L., genes whose depletion causes greater toxicity in KRAS mutant cells S. Shahamatdar, D.H., and S. Swatkoski performed research; S.-C.W., S. Swatkoski, P.M., compared with KRAS WT cells. Surprisingly, many of these genes S.G., M.G., C.M.A., C.C.H., J.-C.S., and G.G. contributed new reagents/analytic tools; do not directly partake in the Ras signaling network, but rather, they M.-T.W., J.-H.L., A.J.S., M.K.H.K, C.J.C., M.J.E., G.G., and J.L. analyzed the data; and act to maintain cell viability by alleviating the stress phenotypes in M.-T.W., J.H.L., and J.L. wrote the paper. cancer cells. We, therefore, proposed the concept of nononcogene The authors declare no conflict of interest. addiction to explain the heightened dependency of Ras mutant cells This article is a PNAS Direct Submission. on stress relief pathways for survival (7). Freely available online through the PNAS open access option. In the aforementioned screen, we identified genetic interactions 1To whom correspondence should be addressed. E-mail: [email protected]. between mutant KRAS and a network of mitotic genes, including This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the mitotic kinase polo-like kinase 1 (PLK1) and the E3 ligase 1073/pnas.1207673110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1207673110 PNAS Early Edition | 1of9 Downloaded by guest on September 26, 2021 − wide RNAi screen (6). ERH is a protein with 104 aa, and its breviated as KRAS mutant line, and the KRAS /wt derivative molecular function is poorly understood. To validate the genetic cells are abbreviated as the KRAS WT line. We found that, in interaction between ERH and the KRAS oncogene, we first each isogenic pair, the KRAS mutant cells showed less viability tested an shRNA targeting ERH that scored in the screen using on ERH knockdown compared with their respective KRAS WT DLD-1 and HCT116 isogenic cells that are either WT or mutant counterpart (Fig. 1A). Western blot and quantitative RT-PCR for KRAS. These isogenic cells were derived by targeted deletion (RT-qPCR) revealed a partial knockdown of ERH by this of the mutant KRASG13D allele (6, 19, 20). For simplicity, the shRNA (Fig. 1B and Fig. S1A). Because this shRNA is the only parental cell line with KRASG13D/wt genotype is hereafter ab- effective shRNA against ERH in our library, we identified two

Fig. 1. Synthetic lethal interactions between ERH and the KRAS oncogene. (A) DLD-1 and HCT116 KRAS mutant cells show less viability compared with their respective WT control after retroviral ERH shRNA infection. Cell viability was assessed 4 d post-shRNA infection (error bars indicated SD of three independent experiments in all figures unless otherwise indicated). (B) Confirmation of ERH protein knockdown by ERH shRNA at 4 d post-shRNA infection. The number below each band indicates relative ERH protein level. (C) DLD-1 and HCT116 KRAS mutant cells show less viability compared with their respective KRAS WT control after ERH siRNA transfection. Cell viability was assessed 3 d post-siRNA transfection (assessment was the same for all siRNA viability experiment unless otherwise stated). (D) Confirmation of ERH protein knockdown by ERH siRNAs at 3 d post-siRNA transfection. (E) An HA-ERH cDNA rescue construct lacking the UTR regions of endogenous ERH remains sensitive to siERH-3, but it is resistant to siERH-5, which was confirmed by Western blot. (F) Stable expression of HA-ERH rescues the toxicity of siERH 5 in both DLD-1 and HCT116 KRAS mutant cells. (G) ERH depletion strongly decreases the ability of DLD-1 KRAS mutant cells to form colonies in soft agarose. Cells were transfected with indicated siRNAs and plated in soft agarose. Colonies were counted 14 d later. (H)Correlationof sensitivities to KRAS and ERH siRNAs in a panel of KRAS mutant (SW1116, SW620, SW403, LS123, and LOVO) and WT (RKO, CACO2, and SW48) CRC cell lines.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1207673110 Weng et al. Downloaded by guest on September 26, 2021 additional ERH siRNAs: one targeting its coding sequence their viabilities (Fig. S1F). Thus, ERH synthetic lethality is selec- PNAS PLUS (siERH-3) and one targeting its 3′-UTR (siERH-5). Transfection tive for the KRAS oncogene but not the PIK3CA oncogene. of these siRNAs in these cell lines, as shown in Fig. 1C and Fig. To assess whether ERH is important for the survival of ad- S1B, also decreased the viability of the KRAS mutant cells more ditional KRAS mutant CRC cell lines, we tested eight CRC cell than the viability of the KRAS WT cells. Furthermore, on ERH lines for their sensitivity to ERH and KRAS depletion. These depletion, we detected higher caspase activity in KRAS mutant lines include five KRAS mutant cell lines (SW1116, SW620, cells compared with KRAS WT cells (Fig. S1C). Western blotting SW403, LS123, and LOVO) and three KRAS WT cell lines and RT-qPCR confirmed that ERH protein was efficiently de- (RKO, CACO2, and SW48). We found that four of five KRAS pleted by both siRNAs (Fig. 1D and Fig. S1D). To ensure that mutant lines are variably sensitive to KRAS knockdown, whereas cell viability change on ERH knockdown is an on-target effect, all three KRAS WT lines are resistant. This finding is consistent we constructed a C-terminal hemagglutinin (HA)-FLAG–tagged with previous findings that KRAS mutant cell lines exhibit differ- ERH cDNA and stably expressed it in DLD-1 and HCT116 cells. ent degrees of KRAS dependency (22). We observed a strong Because this cDNA construct lacks a 3′-UTR, it is resistant to correlation between these cells’ sensitivity to KRAS depletion and knockdown by siERH-5, but it remains sensitive to siERH-3 their sensitivity to ERH depletion (Fig. 1H and Fig. S1G). Thus, (Fig. 1E). As expected, the HA-ERH construct rescued the le- dependency on ERH is not confined within the isogenic KRAS cell thality of siERH-5 but not siERH-3 (Fig. 1F). lines, but it is evident in KRAS-dependent CRC cells in general. To test whether ERH is required for the transformed phenotype of the KRAS mutant cells, we tested ERH-depleted DLD-1 KRAS ERH Regulates Chromosome Congression. ERH is highly conserved mutant in soft agarose colony assay. Loss of ERH nearly com- through , and it shares little with pletely inhibited the anchorage-independent growth of these cells other proteins (Fig. S2A). ERH depletion does not seem to af- (Fig. 1G and Fig. S1E). We next tested whether the synthetic fect the level of KRAS protein or signaling through the MAPK lethality of ERH depletion is selective for the KRAS oncogene. We pathway (Fig. S2B). Because a number of mitotic genes have been depleted ERH in isogenic phosphatidylinositol 3-kinase (PI3K) identified in our previous screen (6), we investigated whether ERH mutant and WT DLD-1 cells (21) and observed no difference in might play a role in cell cycle regulation. Indeed, ERH knockdown CELL BIOLOGY

Fig. 2. ERH is required for chromosome congression. (A) Depletion of ERH leads to accumulation of G2/M cells. Cells were fixed 3 d post-siRNA transfection and stained with propidium iodide to quantify the fraction of G2/M cells (*P < 0.05). (B) IF staining of mitotic cells using phospho-H3 serine 10 (pH3S10) antibody, tubulin antibody, and DAPI revealed that ERH depletion leads to chromosome lagging at spindle pole (white arrowheads). Cells were imaged 3 d post-siRNA transfection. (Scale bar: 10 μm in all images unless otherwise indicated.) (C) Quantification of chromosome congression defects in DLD-1 and HCT116 cells as seen in B.(D) Live-cell video microscopy of U2OS cells stably expressing GFP-H2B undergoing mitosis with or without ERH depletion. Rep- resentative time frames are shown. Cells were imaged 3 d post-siRNA transfection for up to 12 h at 37 °C in an environment chamber. (E) Quantification of average mitosis duration of U2OS GFP-H2B cells as imaged in D. The mitosis duration is defined as the time lapse between nuclear envelop breakdown and anaphase onset. Numbers at the base of the graph indicate total mitotic events measured for each treatment.

Weng et al. PNAS Early Edition | 3of9 Downloaded by guest on September 26, 2021 causes a more pronounced G2/M arrest in KRAS mutant cells investigated whether ERH depletion would influence CENP-E compared with KRAS WT cells (Fig. 2A and Fig. S2C). We next activity. During mitosis, CENP-E localizes to kinetochores [as used immunofluorescence (IF) staining of serine-10 phosphory- marked by the inner protein anticentromere antibody lated H3 to visualize mitotic cells to investigate the po- (ACA)]. Kinetochore localization of CENP-E was dramatically tential cause for the mitotic delay. ERH-depleted cells failed reduced on ERH depletion and restored on HA-ERH rescue to align chromosomes at the metaphase midplate, and lagging (Fig. 3B). To better quantify the localization defect of CENP-E, chromosomes at spindle poles are abundantly evident (Fig. 2 B we arrested cells in prometaphase with nocodazole (Fig. S2D) and C). Importantly, these mitotic defects can be rescued by the and scored the fraction of cells with or without visible CENP-E siRNA-resistant HA-ERH cDNA. To better understand the ef- at kinetochores. ERH depletion resulted in a 60–70% decrease fect of ERH on chromosome congression, we depleted ERH in in the number of cells with CENP-E at kinetochores (Fig. 3C). U2OS cells stably expressing GFP-H2B protein and tracked Surprisingly, ERH depletion causes a dramatic loss of both mitosis using live-cell fluorescence video microscopy. The time CENP-E protein (Fig. 3D) and CENP-E mRNA (Fig. 3E), which duration from nuclear envelope breakdown to anaphase onset in explains the loss of CENP-E at kinetochores. ERH-depleted cells was approximately sixfold longer than the Recruitment of CENP-E to kinetochores depends on the spindle time duration of control cells, reflecting difficulties in chromo- assembly checkpoint (SAC) proteins, and RNAi depletion study some alignment in the absence of ERH (Fig. 2 D and E and indicates that localization of Bub1 to kinetochores in early pro- Movies S1, S2, and S3). phase is necessary for the kinetochore localization of BubR1, We next investigated how ERH might regulate chromosome CENP-E, and MAD2 (23, 24). We, therefore, investigated whether congression at the molecular level. In cells, ERH is Bub1, BubR1 and MAD2 localization is also impaired by ERH distributed throughout the nucleus, but it seems to be excluded depletion. All three proteins localized to the kinetochore normally from the nucleoli. In mitotic cells, ERH is distributed throughout (Fig. 3F), and show normal protein levels in ERH-depleted cells the cells, but it seems to be excluded from . Inter- (Fig. 3G). Furthermore, depletion of ERH did not impair the cell’s estingly, unlike many spindle checkpoint proteins, ERH does ability to stably arrest in prometaphase in the presence of noco- not localize to kinetochores (Fig. 3A). We noticed that the chro- dazole (Fig. S2D), suggesting that the SAC remains intact in mosome congression defects associated with ERH depletion ERHp-depleted cells. Thus, ERH is required for the expression of resemble the defects of CENP-E loss (13, 14). We, therefore, CENP-E but not other SAC proteins.

Fig. 3. ERH is required for the expression of CENP-E. (A) Localization of ERH throughout the cell cycle using IF against the HA-tag in DLD-1 cells stably expressing HA-ERH. (B) ERH depletion causes loss of CENP-E from kinetochore. Asynchronous DLD-1 cells in mitosis were stained with antibodies against CENP-E and ACA (an inner kinetochore protein) with or without ERH depletion and rescue. (C) Quantification of CENP-E localization in DLD-1 and HCT116 cells arrested in prometaphase with ERH depletion and rescue. (D) Western blot reveals loss of CENP-E protein on ERH depletion in asynchronous DLD-1 cells. (E) Real-time qPCR using exon-spanning primers reveals loss of CENP-E mRNA on ERH knockdown and recovery of CENP-E mRNA on rescue. (F) IF staining against Bub1, BubR1, and MAD2 in mitotic cells shows normal localization of these proteins to kinetochores. (G) Western blot shows that the levels of Bub1, BubR1, MAD2, and PLK1 are not affected by ERH depletion.

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1207673110 Weng et al. Downloaded by guest on September 26, 2021 ERH Interacts with SNRPD3 and Is Required for CENP-E mRNA Splicing. protein is the small nuclear ribonucleoprotein Sm D3 (SNRPD3), a PNAS PLUS Because ERH depletion led to loss of CENP-E mRNA, we in- member of the Sm protein complex that is involved in snRNP vestigated how ERH might affect CENP-E expression. ChIP of assembly and pre-mRNA splicing (25, 26). Coimmunoprecipitation HA-tagged ERH protein did not reveal significant enrichment experiments confirmed the interaction between endogenous ERH of ERH at the CENPE promoter (Fig. S3), suggesting that ERH and SNRPD3 in cells (Fig. 4B). Interestingly, siRNA depletion of might not directly regulate CENPE transcription initiation. Be- SNRPD3 also resulted in CENP-E protein loss and chromosome cause the cellular function of ERH is poorly understood, we used congression defect (Fig. 4 C–E). These observations support the stable isotope labeling by amino acids in cell culture MS to iden- model that ERH functions as part of an mRNA splicing complex tify its binding partners (Fig. 4A). One candidate ERH binding that is required for the splicing of CENP-E mRNA. CELL BIOLOGY

Fig. 4. ERH interacts with the splicing factor SNRPD3 and is required for CENP-E mRNA splicing. (A) Schematics of stable isotope labeling by amino acids in cell culture MS to identify ERH interacting proteins. Proteins that immunoprecipitate selectively with HA-ERH have positive light-to-heavy (L/H) amino acid ratios. The top five candidate proteins are shown. (B) Reciprocal immunoprecipitation (IP) experiments in DLD-1 cells show that endogenous SNRPD3 coimmunoprecipitates with HA-tagged ERH and that endogenous ERH coimmunoprecipitates with endogenous SNRPD3. (C) Western blot verifying depletion of SNRPD3 by two independent siRNAs leads to loss of CENP-E protein. Cells were analyzed 2 d post-siRNA transfection. (D) IF shows that depletion of SNRPD3 in DLD-1 cells leads to chromosome congression defects similar to ERH depletion; 2 d post-siRNA transfection, cells were fixed and stained with pH3S10 antibody, tubulin antibody, and DAPI. (E) Quantification of the chromosomal congression defects in DLD-1 cells on SNRPD3 depletion as seen in D.(F) Real- time qPCR using exon-spanning and splice junction primers at three exon–intron junctions of the CENP-E pre-mRNA. The schematic indicates the location of PCR primers and the species of mRNA that they detect (EE, exon–exon PCR; EI, exon–intron PCR; IE, intron–exon PCR). Depletion of ERH leads to a loss of the spliced products (EE amplicons) and an increased in unspliced mRNA (EI and IE amplicons) at each exon–intron junction.

Weng et al. PNAS Early Edition | 5of9 Downloaded by guest on September 26, 2021 Human CENP-E is a very large protein with 2,663 aa that are ERH Regulates the Expression of a Subset of Genes Involved in Cell encoded by 49 exons spanning a 92,604-nt pre-mRNA transcript. Cycle. Because ERH is required for the splicing and expression To test whether ERH is required for CENP-E mRNA splicing, of CENP-E, we asked whether CENP-E depletion would also we used intron-spanning primers and splice junction primers to constitute synthetic lethality with KRAS. Depletion of CENP-E compare the relative levels of CENP-E mature and pre-mRNA in KRAS WT and mutant CRC cell lines, however, showed in cells with or without ERH knockdown. We tested three similar toxicity across all lines, with no clear distinction between A splicing junctions with large intervening introns and three junc- KRAS WT and mutant cells (Fig. S5 ). Thus, CENP-E alone tions with small intervening introns (Fig. 4F and Fig. S4A). Real- does not mediate the synthetic lethal interaction between ERH time qPCR revealed that, on ERH depletion, there was a de- and KRAS. We also tested whether pharmacological inhibition crease in mature splicing products at all six exon–intron junctions, of aurora-B kinase, which is required for both CENP-E activity with a concomitant increase in unspliced mRNA (Fig. 4F and Fig. (29) and the SAC (30), might synergize with ERH siRNAs. Two structurally distinct aurora-B kinase inhibitors, AZD1152 (31) S4B). These results suggest that ERH is required for CENP-E and ZM447439 (30), exhibited modest selectivity against KRAS pre-mRNA splicing. Consistent with our finding that ERH mutant cells but were not synergistic with ERH siRNAs (Fig. interacts with the Sm complex subunit SNRPD3, siRNA de- S5B). Thus, the nature of mitotic stress in KRAS mutant cells is pletion of two other Sm complex subunits SNRPD1 and SNRPD2 C D likely to be complex. also led to the loss of CENP-E mRNA (Fig. S4 and ). Mis- We next carried out gene expression profiling in DLD-1 cells to spliced mRNAs are often rapidly removed in cell through the identify additional genes that might be regulated by ERH. To nonsense-mediated mRNA decay (NMD) pathway (27). Half-life distinguish siRNA on-target effects from off-target effects, we measurement of spliced CENP-E mRNA in the presence of the compared gene expression profiles of DLD-1 cells transfected with transcription inhibitor actinomycin D indicates that ERH de- siERH-5 with the profiles of control siRNA-transfected cells and pletion did not increase the degradation rate of spliced CENP-E cells transfected with siERH-5 together with HA-ERH rescue mRNA (Fig. S4E). siRNA depletion of up-frameshift suppressor (Fig. 5A and Fig. S6A). We define on-target genes as those genes 1 homolog (UPF1), a key protein in the NMD pathway (28), with expression that is altered by siERH-5 but restored to normal resulted in a partial rescue of CENP-E mRNA and a further level on HA-ERH rescue. This analysis identified 64 genes that are increase in CENP-E pre-mRNA levels (Fig. S4F). Thus, the down-regulated and 23 genes that are up-regulated on ERH de- NMD pathway plays a role in degrading aberrantly spliced or pletion. Ingenuity analysis revealed a significant enrichment for unspliced CENP-E pre-mRNA in the absence of ERH. genes involved in cell cycle and DNA replication/repair pathways

Fig. 5. ERH regulates the expression of multiple cell cycle genes. (A) Gene expression profiling comparison of DLD-1 cells transfected with siNEG, siERH-5, and siERH-5 plus HA-ERH rescue identified 87 genes with expressions that are regulated by ERH. (B) Ingenuity Pathway Analysis identified cell cycle and DNA replication/repair as the top two gene networks regulated by ERH. (C) The ERH gene expression signature and two KRAS signatures (Singh KRAS dependency signature and Bhattacharjee lung KRAS signature) were used to score colon and lung cancer cell lines [Cancer Cell Line Encyclopedia (CCLE) dataset] and colon [The Cancer Genome Atlas (TCGA) dataset] and lung (combined Tomida and Chitale dataset) tumor samples. Within each dataset, the ERH signature neg- atively correlated with the KRAS signature. (D) A model of the function of ERH in mRNA splicing. Our data support the notion that ERH is required for the mRNA splicing of CENP-E and potentially other cell cycle genes and therefore, their expression, and KRAS mutant cells are more dependent on the function of ERH for survival. Dashed lines indicate functional connections with unclear molecular steps that warrant additional investigation.

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1207673110 Weng et al. Downloaded by guest on September 26, 2021 that are dependent on ERH for their expression (Fig. 5B and ERH Expression Is Associated with CRC Patient Survival. Our in vitro PNAS PLUS Fig. S6B). These genes include CENPE, the condensin subunit analysis in CRC cell lines shows that KRAS mutant CRC cancer SMC4, the mitotic kinesin and cancer testis antigen KIF20B,and cells are more dependent on ERH expression for survival. This DNA damage repair genes ATR, MRE11A, RAD50, RAD54B, and finding, in turn, suggests that ERH gene expression might be FANCM. Thus, ERH is likely to regulate cell cycle genes, and its associated with malignancy of KRAS mutant tumors in patients. synthetic lethal effect could be caused by the simultaneous de- To evaluate this hypothesis, we analyzed the association between regulation of multiple genes. ERH mRNA expression and patient survival in two independent We next tested whether an ERH gene expression signature CRC patient cohorts at the National Taiwan University Hospital consisting of all 87 genes that are deregulated by ERH depletion (NTUH cohort) and the National Cancer Institute (NCI cohort). negatively correlates with two KRAS signatures: the Singh KRAS The patient characteristics in these two cohorts are shown in fi dependency signature (22) and the Bhattacharjee lung KRAS Table S1. ERH expression is not signi cantly different between A B signature (6). We first analyzed CRC and lung cancer cell lines KRAS mutant and WT tumors (Fig. 6 and ). However, whereas ERH expression was not associated with survival of that were recently characterized by the Cancer Cell Line Ency- C D clopedia project (32). Cell lines were independently scored using patients with KRAS WT tumors (Fig. 6 and ), in patients with KRAS mutant tumors, low ERH expression was associated each of the three signatures, and the correlations across the with significantly better survival (Fig. 6 E and F). signatures were analyzed. Consistent with our hypothesis, ERH We also evaluated the clinical relevance of ERH expression in signature scores show a moderate but consistent negative cor- stage I/II lung adenocarcinoma using a publicly available gene relation with the Singh KRAS dependency signature scores. In expression dataset from the Gene Expression Omnibus database the lung cancer cell lines, ERH signature scores also negatively (accession no. GSE31210) (34). In this cohort, ERH expression correlated with the Bhattacharjee lung KRAS signature scores was higher in tumors with EGF receptor (EGFR) mutations C C D (Fig. 5 and Fig. S6 and ). We next carried out a similar (Fig. S7A). In the subset of patients with KRAS mutant tumors, analysis in a collection of CRC tumor samples characterized by we again observed a strong trend in favor of longer survival with The Cancer Genome Atlas (the TCGA colorectal tumors) (33) lower levels of tumor ERH expression, although the P value just and a combined set of lung tumor samples (the Tomida_Chitale failed to reach significance (Fig. S7B). Importantly, ERH ex- lung tumors) (1, 3). We again observed a modest but consistent pression was unrelated to prognosis in patients with EGFR

negative correlation between the ERH and the Singh and Bhat- mutations or patients whose tumors were WT for both EGFR CELL BIOLOGY tacharjee signature scores in these samples (Fig. 5C and Fig. S6 E and KRAS (Fig. S7 C and D). and F). Taken together, our data support the model that KRAS Although the number of patients was relatively small in each mutant cells are more dependent on ERH and the gene expres- of the three cohorts analyzed, our results are highly consistent sion program ERH regulates for survival (Fig. 5D). across cohorts, and they support the model where KRAS mutant

Fig. 6. Clinical association between ERH expression and survival in CRC patients with KRAS mutation tumors. (A and B) Tumor ERH expression in the NTUH and NCI cohorts. No difference in ERH expression was observed between KRAS WT and mutant tumors in either cohort. (C and D) In the subset of patients with KRAS WT tumors, ERH expression is not associated with survival. Patients were divided into ERH low and high groups based on median ERH expression, and their survival was plotting using the Kaplan–Meier analysis (uptick indicates censoring events). (E and F) In the subset of patients with KRAS mutant tumors, low ERH expression is significantly associated with better survival.

Weng et al. PNAS Early Edition | 7of9 Downloaded by guest on September 26, 2021 cancer cells are more dependent on ERH for their viability and is more conserved with metazoans (51). Furthermore, during mi- low ERH expression could serve to constrain the malignancy of tosis, chromosome congression occurs in S. pombe (52) but not KRAS mutant colorectal and lung cancers. S. cerevisiae (53). It is, therefore, an intriguing possibility that ERH might have coevolved with CENP-E–like to Discussion regulate chromosome congression. Our microarray analysis KRAS is a particularly potent oncogene, because it can activate indicates that, in addition to CENPE, ERH is also required for the a number of downstream effector pathways, such as the MAPK expression of additional cell cycle genes, including SMC4, ATR,and pathway, the PI3K pathway, and the Ral GTPases (2, 4). Cancer MRE11A. Whether ERH is also required for the splicing of these cells that are mutant in the KRAS gene exhibit the classical genes’ pre-mRNA requires additional investigation. phenotype of oncogene addiction, which is reflected by their This study, together with previous work by us and others (6, dependence on mutant KRAS for their transformed phenotype 35), supports the notion that KRAS mutant cells experience el- and survival (19, 22) (Fig. 1H). However, dissecting the down- evated mitotic stress and therefore, are more dependent on the stream genetic dependencies that constitute Ras addiction has proper function of multiple mitotic genes. Our finding is con- proved more complex. Several genome-wide screens against sistent with studies showing that the expression of mutant Ras, the KRAS oncogene have identified a surprisingly diverse set of either exogenously or from the endogenous , can induce genes whose depletion causes greater toxicity in KRAS mutant in mouse fibroblasts (40, 54, 55). Downstream of Ras, cells compared with KRAS WT cells (6, 35–39). We think that the MAPK pathway has been implicated in this process (56, 57), this finding reflects a broad dependency of Ras mutant cells on although the molecular steps connecting Ras with genomic in- various stress relief pathways that constitute what we termed stability remain unclear and warrant additional investigation. nononcogene addictions (7). Importantly, we did not observe synthetic lethality between ERH In this study, we characterized an Ras synthetic lethal gene and the PIK3CA oncogene. Furthermore, ERH expression is ERH and provided evidence that ERH is critical for the ex- inversely correlated with survival of patients whose tumors har- pression of the mitotic motor protein CENP-E and consequently, bor KRAS mutations, but it is not associated with survival in chromosome congression. ERH interacts with the Sm protein those patients whose tumors are WT for KRAS or harbor EGFR SNRPD3 and is required for the splicing of CENP-E mRNA. mutations. Thus, dependency on ERH is likely a nononcogene Gene expression microarray revealed that siRNA depletion of addiction that is unique to KRAS-transformed tumor cells. ERH results in the loss of expression of many cell cycle and Understanding the mechanisms underlying this addiction could DNA replication/repair genes. The synthetic lethality between help identify new avenues of therapeutic approaches for Ras- ERH and KRAS, thus, is likely to reflect the increased cell cycle driven tumors, and our study suggests targeted disruption of the stress in the KRAS mutant cells, which was documented pre- mRNA splicing machinery could present one such potential av- viously by us and others (6, 40). enue for therapeutic exploration. ERH is a highly conserved protein among metazoans and plants (Fig. S2A). Amino acid sequence similarity of ERH or- Materials and Methods thologs between human and Dictyostelium is 80%, and amino Cell lines and Reagents. The KRAS mutant and WT isogenic DLD-1 and HCT116 acid sequence similarity of ERH orthologs between human and cell lines were gifts from Bert Vogelstein (Johns Hopkins University School of Arabidopsis is 78%. Distant ERH orthologs exist in the fission Medicine, Baltimore, MD) and have been described previously (6, 19, 20). All yeasts Schizosaccharomyces pombe and S. japonicus but not in the CRC cell lines were maintained in McCoy’s 5A media with 10% fetal bovine budding yeast Saccharomyces cerevisiae. Such a high degree of serum. ERH shRNA was expressed using the MSCV-PM retroviral vector. All conservation suggests that ERH plays an important role in cel- siRNAs were from Qiagen and Dharmacon and their sequence information is lular function. ERH has a unique protein fold (41) and has been provided in SI Materials and Methods. Rabbit polyclonal anti-ERH antibody – was generated using the peptide sequence QPTKRPEGRTYADYC (GenScript). implicated to play a role in nuclear gene expression (16 18) and MAD2 and BubR1 antibodies were gifts from Stephen Taylor (University of cell growth (42, 43). Our study indicates that, through its in- Manchester, Manchester, United Kingdom). All other antibodies were pur- teraction with SNRPD3, ERH is likely to function as a subunit of chased from commercial sources as listed in SI Materials and Methods. an mRNA splicing complex. This finding is consistent with a re- cent proteomics study identifying ERH as an interactome mem- MS and Microarray Gene Expression Analysis. The method of stable isotope ber of the survival of motor neuron complex that is required for labeling by amino acids in cell culture (SILAC) was used to label cells prior to spliceosome assembly (44). Although mRNA splicing factors are affinity purification of HA-tagged ERH and its associated proteins from total often inferred to play a housekeeping role, our data indicate that cell lysates. Following trypsin digestion, peptide ions were detected in a data- ERH depletion has a striking effect on chromosome congression dependent manner using an LTQOrbitrap Velos (Thermo Electron). The six through its selective regulation of CENP-E mRNA splicing. most intense precursor ions from each full MS1 scan were selected for MS/MS CENP-E is degraded on mitotic exit (12), and thus, its protein by collision-induced dissociation. LC MS/MS data were searched using the MASCOT algorithm within Proteome Discoverer 1.2 (Thermo Electron). Gene levels are sensitive to perturbation in its mRNA level. Several expression profile was obtained using GeneChip U133 Plus large-scale RNAi studies have implicated the role of mRNA – 2.0 Array (Affymetrix). GeneSpring software 11.5 (Agilent) was used for micro- splicing factors in mitosis (45 47). These factors include members array data analysis. For gene expression signature analysis, a two-sided t statistic of the U1, U2, and U5 U4/6 snRNPs and some SR proteins. The comparing the average of the signature up genes with the average of the sig- mechanisms by which these splicing factors affect mitosis, how- nature down genes within each cell line/tumor was used to compute the gene ever, are not known. Recently, the ubiquitin specific peptidase signature score. Signature scores were correlated using the Pearson method. 4 (USP4) was found to be required for the splicing of Bub1 (48), whereas depletion of the serine/arginine-rich splicing factor SON Clinical Samples, Patient Data Analysis, and Ethics Statement. Patient studies causes pleotropic mitotic defects (49). ERH is likely to operate were approved by the Ethical Committee of the National Taiwan University in a distinct pathway, because it is required for the splicing of Hospital (NTUH cohort) and the Institutional Review Board of the National CENP-E pre-mRNA for the expression of Bub1, BubR1, MAD2, Institutes of Health and the Institutional Review Board for Human Subject Research at the University of Maryland (NCI cohort), respectively. Written or PLK1. Accordingly, the ERH-depleted cells exhibit defective Cenpe informed consent was received from all participants before inclusion in the chromosomal congression but intact SAC, similar to -null study. Somatic mutation status in KRAS was determined by Sanger se- mouse embryonic fibroblasts (50). Interestingly, we were able to quencing. ERH gene expression was determined by quantitative RT-PCR identify distant ERH orthologs in fission yeast but not budding (NTUH cohort) or by microarrays (NCI cohort). yeast. S. pombe has considerably more introns in its genome A full description of the materials and methods used for this work is compared with S. cerevisiae, and the S. pombe splicing machinery described in SI Materials and Methods.

8of9 | www.pnas.org/cgi/doi/10.1073/pnas.1207673110 Weng et al. Downloaded by guest on September 26, 2021 ACKNOWLEDGMENTS. We thank Drs. , Tom Misteli, Mary a Liver Disease Prevention and Treatment Research Foundation (Taiwan) PNAS PLUS Dasso, and Lewis Cantley for critical reading and comments on this manu- Fellowship (to M.-T.W.) and US National Cancer Institute Center for script. We thank Dr. Stephen Taylor for antibodies and Drs. Bert Vogelstein, Canter Research (NCI-CCR) Intramural Research Program ZIA BC 011259 Thomas Ried, and Mary Dasso for cell lines. This work is supported by (to J.L.).

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