Targeting Calcium Signaling Induces Epigenetic Reactivation of Tumor Suppressor Genes in Cancer Noel€ J.-M

Published OnlineFirst December 30, 2015; DOI: 10.1158/0008-5472.CAN-14-2391

Cancer Therapeutics, Targets, and Chemical Biology Research

Targeting Calcium Signaling Induces Epigenetic Reactivation of Tumor Suppressor Genes in Cancer Noel€ J.-M. Raynal1,2, Justin T. Lee1, Youjun Wang3, Annie Beaudry2, Priyanka Madireddi1, Judith Garriga1, Gabriel G. Malouf4, Sarah Dumont4, Elisha J. Dettman4, Vazganush Gharibyan4, Saira Ahmed4, Woonbok Chung1, Wayne E. Childers5, Magid Abou-Gharbia5, Ryan A. Henry6, Andrew J. Andrews6, Jaroslav Jelinek1, Ying Cui7, Stephen B. Baylin7, Donald L. Gill8, and Jean-Pierre J. Issa1

Abstract

Targeting epigenetic pathways is a promising approach for prominently cardiac glycosides, did not change DNA methyl- cancer therapy. Here, we report on the unexpected finding that ation locally or histone modifications globally. Instead, all 11 targeting calcium signaling can reverse epigenetic silencing of drugs altered calcium signaling and triggered calcium-calmod- tumor suppressor genes (TSG). In a screen for drugs that ulin kinase (CamK) activity, leading to MeCP2 nuclear exclu- reactivate silenced gene expression in colon cancer cells, we sion. Blocking CamK activity abolished gene reactivation and found three classical epigenetic targeted drugs (DNA methyl- cancer cell killing by these drugs, showing that triggering ation and histone deacetylase inhibitors) and 11 other drugs calcium fluxes is an essential component of their epigenetic that induced methylated and silenced CpG island promoters mechanism of action. Our data identify calcium signaling as a driving a reporter gene (GFP) as well as endogenous TSGs in new pathway that can be targeted to reactivate TSGs in cancer. multiple cancer cell lines. These newly identified drugs, most Cancer Res; 76(6); 1494–505. 2015 AACR.

Introduction novo methylation in cytosine of CpG dinucleotide at the promoter region of TSG results in stable gene silencing through direct In cancer, the epigenome is aberrantly reprogrammed, leading inhibition of transcription factor binding or by recruitment of to a wide range of heritable changes in gene expression such as methyl-binding domain (MBD) proteins such as MeCP2 (1, 2). silencing of tumor suppressor genes (TSG; ref. 1). The most These MBDs are associated with other repressor complexes includ- studied epigenetic aberrations in cancer involve DNA methylation ing histone deacetylases (HDAC) that are responsible for global and histone posttranslational modifications. Acquisition of de loss of histone acetylation, resulting in gene silencing and het- erochromatin formation (2). As these epigenetic modifications are reversible, one goal of 1Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania. epigenetic therapy of cancer is to reverse these alterations and 2Departement de pharmacologie, Universite de Montreal and induce TSG reactivation, leading to cancer cell differentiation and Sainte-Justine University Hospital Research Center, Montreal,Qu ebec, cancer cell death (3). Clinical efficacy of epigenetic drugs led to 3 Canada. Beijing Key Laboratory of Gene Resources and Molecular their approval for the treatment of hematologic malignancies and Development College of Life Sciences, Beijing Normal University, Beijing, P.R. China. 4Department of Leukemia, The University of Texas occasional proof-of-principle responses can be seen in solid MD Anderson Cancer Center, Houston, Texas. 5Moulder Center for tumors (2, 4). However, treatment options are limited to a small 6 Drug Discovery Research, Philadelphia, Pennsylvania. Department number of epigenetic drugs approved in the clinic with two DNA of Cancer Biology, Fox Chase Cancer Center, Philadelphia, Pennsyl- vania. 7The Sidney Kimmel Comprehensive Cancer Center at Johns methylation inhibitors (decitabine and azacitidine) and two Hopkins, Baltimore, Maryland. 8Department of Cellular and Molecular HDAC inhibitors (vorinostat and depsipeptide). There is a need Physiology, The Pennsylvania State University College of Medicine, to discover new candidate epigenetic drugs, including some that The Milton S. Hershey Medical Center, Hershey, Pennsylvania. work through other mechanisms of action. Drug discovery initia- Note: Supplementary data for this article are available at Cancer Research tives are underway in rare and specific cancer types with well- Online (http://cancerres.aacrjournals.org/). defined mutations in epigenetic effectors. However, these efforts Current address for G.G. Malouf: G.G. Malouf, Hopital^ de la Pitie-Salp etri ere,^ may take years before approval and may have limited effects Medical Oncology, Paris, France. outside of a restricted patient population (5). Corresponding Author: Jean-Pierre J. Issa, Fels Institute for Cancer Research To discover new epigenetic drugs that can be rapidly tested in and Molecular Biology, Temple University School of Medicine, 3307 North Broad the clinic, we performed an unbiased epigenetic drug screen – Street, Rm 154 PAHB, Philadelphia, PA 19140. Phone: 215-707-4307; using FDA-approved drug libraries. The rationale is that pos- Fax: 215-707-1454; E-mail: [email protected] itive hits can be rapidly repositioned for cancer treatment doi: 10.1158/0008-5472.CAN-14-2391 because these drugs have known safety, pharmacodynamics, 2015 American Association for Cancer Research. and pharmacokinetics (6). As a platform for epigenetic drug

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Figure 1. Epigenetic drug screening reveals candidate epigenetic drugs among FDA-approved libraries. A, scheme showing CMV-GFP locus in YB5 cells. CMV promoter is DNA hypermethylated and marked by repressive chromatin. Epigenetic drugs induced GFP reactivation as shown by GFP fluorescence after decitabine treatment (50 nmol/L, 72 hours). B, illustration of dose schedules selected for drug screening. C, drug screening results after treatment with library of drugs at 10 mmol/L for 72 hours. Twenty-three positive hits were identified (n 1). D, drug screening results after treatment with the same library at 50 mmol/L for 24 hours. Seventy-seven positive hits were identified (n 1). E, Venn diagram showing the number of positive hits specific or shared to each dose schedule and the validation results. F, positive hits and screening values of GFP-expressing cells (n 1).

screening, we used the well-characterized YB5 cell-based sys- reactivate silenced TSGs, we used the YB5 system as a cell-based tem, which is derived from the human colon cancer cell line, assay for epigenetic drug screening. SW48 (7, 8). YB5 cells contain a single insertion of cytomeg- Here, our screen revealed a new mechanism where targeting alovirus (CMV) promoter driving GFP gene. GFP expression is calcium signaling can reactivate TSGs silenced by epigenetic silenced in >99.9% of YB5 cells by epigenetic mechanisms mechanism in cancer cells. The alteration in calcium signaling because its CMV promoter has DNA hypermethylation and is results in activated calcium-calmodulin kinase (CamK), which embeddedinrepressivechromatinwithhistonedeacetylation played a central role in TSG reactivation and cancer cell killing. and histone methylation marks (Fig. 1A; ref. 7). In YB5 cells, GFP behaves similarly to endogenous TSGs silenced by epige- Materials and Methods netic mechanisms and it can be reactivated by treatment with DNA methylation inhibitors and/or HDAC inhibitors (7, 8). Cell culture We previously demonstrated that GFP reactivation induced by Human colon cancer cell line YB5 and its parental cell line decitabine is characterized by both DNA demethylation and SW48 were cultured in L-15 medium. Human colon cancer cell chromatin resetting at the CMV promoter region (7). Moreover, line HCT116 was cultured in McCoy's medium. In HCT116 we also showed that HDAC inhibitors induced GFP reactivation human colon cancer cells, GFP sequence was inserted in exon 2 through chromatin resetting at the CMV promoter with an of the TSG secreted frizzled-related protein 1 (SFRP1) making the increase in histone acetylation without any changes in DNA HCT116 SFRP1-GFP cell line. This locus is silenced by promoter methylation (8). As the goal of the epigenetic therapy is to DNA hypermethylation in HCT116 (9). GFP expression detected

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by flow cytometry corresponded to the activity of SFRP1 promoter bleaches into GFP channel. Drugs that showed more than and was measured in less than 10% of the cells. Normal colon 8% of the cells in the double positive quadrant were discarded. cells, CRL-1831, were grown in DMEM F-12 medium supplemen- This threshold value was calculated as the sum of the average ted with 10 ng/mL cholera toxin, 0.005 mg/mL insulin, 0.005 plus one SD of the percentage of cells detected in the double mg/mL transferrin, 100 ng/mL hydrocortisone, and 100 mmol/L positive quadrant in the entire screen dataset. Second, the HEPES. Leukemia cell lines, K562 and HL-60, were cultured in lowest detection threshold for GFP reactivation was set at þ RPMI1640. HEK293T were grown in DMEM. All cell culture 2.2% of the cells present in the GFP quadrant. This value was media were supplemented with 10% FBS. Colon cancer and established experimentally and corresponded to the lowest GFP leukemic cells were grown in log phase in 1% and 5% CO2 value obtained in the screen that was confirmed during our atmosphere, respectively. SW48, K562, and HL-60 cell lines were validation step with drugs purchased from independent obtained from ATCC (2005–2007) and were authenticated at MD suppliers. Anderson Cancer Center genomic core facility by DNA finger- printing in 2011. CRL-1831 cell line was obtained from ATCC in RNA extraction, cDNA synthesis, qPCR 2012 and used within 2 months from the purchase. HCT116 and Total RNA (2 mg) was extracted using TRIzol (Invitrogen) and HEK293T were obtained from the laboratories of S.B. Baylin and cDNA was synthesized using High-Capacity cDNA Kit (Applied D.L. Gill and were genetically engineered cell lines that were not Biosystems). Quantification of cDNA was done by qPCR with the further authenticated. Universal PCR Master Mix (Bio-Rad) using ABI Prism 7900HT system. Results were obtained from at least three independent Drugs and treatments experiments where each sample was analyzed in triplicate. 18S Drug libraries of FDA-approved drugs were obtained from NCI- was used as a reference gene. All primers have been described Developmental Therapeutics Program (Combo Plate 3948/99 previously (7, 8). containing 77 drugs, NCI Oncology Drug set 4640/34 containing 52 drugs, NCI Oncology Drug set 4641/34 with 37 drugs) and DNA extraction and DNA methylation analysis from commercially available U.S. Drug collection library with DNA extraction, bisulfite conversion, and pyrosequencing were 1,040 drugs (MS Discovery). A total of 1,118 unique FDA- carried out as previously described (7, 8). approved drugs were screened. Log-phase growing YB5 cells were treated with drug libraries (n 1) using two different schedules. Histone extraction and Western blots The first schedule consisted of a 72-hour treatment at 10 mmol/L For acid histone extraction, cells were harvested and washed where drugs and media were replaced every day. Cells were twice with ice-cold PBS supplemented with 10 mmol/L sodium incubated for an additional 24 hours without drugs before anal- butyrate to retain levels of histone acetylation. Cell pellets were ysis. This schedule was designed to discover putative new hypo- resuspended cells in Triton Extraction Buffer [TEB:PBS containing methylating agents as the induction of DNA demethylation may 0.5% Triton X100 (v/v), 2 mmol/L phenylmethylsulfonylfluor- require several cell divisions to become detectable (2). The second ide, 0.02% (w/v) NaN3, complete protease inhibitor cocktail, 10 schedule consisted of a 24-hour treatment at 50 mmol/L. Cells mmol/L sodium butyrate]. Cell lysis was induced after 10 minutes were immediately analyzed after the treatment. This schedule was incubation on ice with gentle stirring. Lysates were centrifuged at designed to discover drugs that would reactivate GFP by chroma- 6,500 g for 10 minutes at 4 C. Supernatant was discarded. Cells tin remodeling because, in this cell line, HDAC inhibitors can were washed in half the volume of TEB and centrifuged as before. reactivate GFP in 24 hours without any changes in DNA meth- Pellets were resuspended in 0.2 N HCl. Histones were acid ylation at the CMV promoter. For validation purposes and phar- extracted overnight at 4 C. Samples were centrifuged at 6,500 macologic (leptomycin L2913) modulation, drugs were pur- g for 10 minutes at 4 C. Histone proteins contained in the chased at Sigma-Aldrich and dissolved either in DMSO, ethanol, supernatant were saved and protein concentration was deter- or sterile PBS according to the manufacturer's recommendations mined using the Bradford assay. Extracted histones were stored and stocks were kept frozen at 80C. CamK inhibitors were at 80 C. Histone extracts were run in 12% SDS–PAGE precast purchased at EMD Millipore (Kn-93, 422712 and Kn-92, gels (Bio-Rad, 345-0118). Proteins were transferred to polyviny- 422709). lidene difluoride (PVDF) membranes in 10 mmol/L CAPS (pH 11) containing 10% methanol and detected by using specific Flow cytometry primary antibodies and horseradish peroxidase–conjugated sec- After treatment, cells were trypsinized and resuspended in ondary antibodies (GE Healthcare) and Enhanced Chemilumi- cell culture media with propidium iodide (PI) to stain dead nescence reagent (Pierce). PVDF membranes were incubated with cells. For epigenetic drug screening with YB5 cells, GFP and PI specific primary antibodies. Commercially available antibodies fluorescence were measured using BD LSR II flow cytometer. were purchased for H3K4-3me (Active motif, 39159), H3K9-3me Validations were performed using Millipore Guava flow cyt- (Abcam, AB 8898), H3K14-Ac (Active motif, 39697), H3K9-Ac ometer with YB5 or SFRP1-GFP HCT116 cells. Flow cytometry (Active motif, 39917), H3K27-3me (Active motif, 39155), analysis was also used prior to any type of extractions (RNA, H3K27-Ac (Upstate Biotechnology, 07360), H3 (Active motif, DNA, and proteins) to ensure a similar level of GFP reactivation 39763), and MeCP2 (Abcam, AB2828). following drug treatments for each biologic replicate. Flow cytometry results were plotted as GFP fluorescence on the x- Statistical analysis axis and PI on the y-axis (Supplementary Fig. S1A–S1D). Differences between groups were assessed using a one-way Positive hits were designated using two selection criteria. First, ANOVA. The P value was evaluated by the Tukey–Kramer multiple we excluded all autofluorescent drugs (like antimalarials) comparison test. Statistics and graphical representations were because autofluorescence creates a false positive signal that performed using GraphPad Prism software.

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Results endogenous TSGs silenced by epigenetic mechanism in YB5 cells (CDH13, RIL, TIMP-3) as well as the stress response gene P21 Epigenetic drug screening and positive hits validation (CDKN1A; Supplementary Fig. S3A–S3D). In addition, we mea- We screened over 1,100 FDA-approved drugs obtained from sured SFRP1 reactivation, another DNA hypermethylated TSG in the NCI and commercial sources using two dose schedules (Fig. HCT116 colon cancer cells (9, 10). In this cell line, GFP sequence 1B). The first schedule (10 mmol/L for 72 hours followed by 24 was inserted in exon 2 of SFRP1 making its activation detectable hours without drug) was designed to discover epigenetic drugs by flow cytometry (9). Similar to YB5 cells, all drugs reactivated that require several cell cycles to induce robust gene reactiva- SFRP1 gene in the HCT116 SFRP1-GFP system (Fig. 3B). To tion, as observed with DNA methylation inhibitors. This sched- identify a mechanism of action, we asked whether candidate ule induced significant DNA demethylation and was associated epigenetic drugs induced gene reactivation through known epi- with gene reactivation in YB5 cells (7). The second schedule (50 genetic mechanism such as DNA demethylation or histone acet- mmol/L for 24 hours) was selected to discover epigenetic drugs ylation. First, we measured DNA methylation locally in the that produce rapid gene reactivation, as seen with HDAC promoter regions of reactivated genes (CMV and endogenous inhibitors. On the basis of the very low background in the TSGs) and at LINE-1 repeats to assess global DNA methylation YB5 system (less than 0.1% of the cells express detectable levels levels. Using bisulfite pyrosequencing assays designed in the of GFP; Supplementary Fig. S1A) and activity seen in negative promoter regions close to the transcription start site (8), we found controls, positive hits in the screen were defined as those non- þ that none of the drugs reduced DNA methylation at the CMV (Fig. autofluorescent drugs inducing more than 2.2% of GFP cells 3C) or TIMP-3 (Supplementary Fig. S4A) promoters or globally at (Supplementary Fig. S1B–S1E). LINE-1 repeats (Fig. 3D), except for decitabine (a well-known The drug screen revealed 23 (2%) and 77 (7%) positive hits at hypomethylating drug). We then measured quantitatively histone 10 mmol/L (for 72 hours) and at 50 mmol/L (for 24 hours) acetylation levels by mass spectrometry on 9 residues in histone 3 respectively, whereas 12 drugs (1%) were identified in both (H3) and histone 4 (H4). No significant changes were observed, schedules (Fig. 1C–E). The highest inducers of GFP expression except with vorinostat, a well-known HDAC inhibitor that were the DNA methylation inhibitors decitabine (at 10 mmol/L, increased histone acetylation levels on lysine 9, 14, 18, and 23 for 72 hours) and azacitidine (at 50 mmol/L, for 24 hours) þ of histone 3 and on lysine 8, 12, and 16 of histone H4 (Fig. 3E and producing, respectively, 30% and 16% GFP cells (Supplemen- Supplementary Fig. S4B). A similar lack of global effects was tary Tables S1 and S2). Azacitidine induced GFP reactivation after observed by Western blots for histone-activating marks (H3K4- a short treatment of 24 hours, which can be explained by the me3, H3K9-ac, and H3K27-ac) or histone-inactivating marks number of YB5 cells in S-phase during the treatment (2). The (H3K9-me3 and H3K27-me3; Supplementary Fig. S4C). HDAC inhibitor vorinostat was also identified in this screen þ þ producing 2.6% GFP cells (Supplementary Table S2). Thus, all Intracellular Ca2 fluxes induced by the newly identified drugs three known epigenetic drugs represented in the libraries were As neither DNA demethylation nor global chromatin modifi- rediscovered in this blinded screen, validating the approach. cations explained TSG reactivation by the newly identified drugs, Because of the unexpected large number of positive hits, we we searched for alternative mechanisms. Surprisingly, when fi focused on the 12 drugs identi ed in both dose schedules (Fig. 1E). searching for a mechanism of action of the newly identified drugs, For validation, compounds were purchased from separate suppliers we noted that most of the (9/11) drugs were known to increase þ and tested for GFP reactivation in YB5 cells. Of these 12 hits, all intracellular calcium (Ca2 ) levels (11–14). No data were found except tilmicosin (which was low positive) were validated. These 11 regarding the three known epigenetic drugs and calcium signaling. fi validated drugs fell into three groups (Fig. 1F). The rst one Interestingly, it was reported in neurons that cytosolic increase in þ included the known epigenetic drugs decitabine, azacitidine, and Ca2 levels activate CamK, which releases the methyl-binding vorinostat. The second group encompassed cardiac glycosides such protein MeCP2 from silenced promoters, leading to gene reacti- as ouabain, lanatoside C, digoxin, digitoxin, and proscillaridin A. vation (15, 16). All cardiac glycosides represented in the libraries reactivated GFP, To test whether this mechanism could explain TSGs reactiva- suggesting a drug class effect. The last group was labeled as anti- tion in cancer cells by the newly identified drugs, we first char- fi þ þ biotics/others and contained oxyquinoline (antibiotic), disul ram acterized Ca2 channels in YB5 cells. Using Ca2 sensitive dye (a drug used for alcoholism treatment), and arsenic trioxide (anti- (fura-2), we determined that YB5 cells do not express functional þ cancer drug; Fig. 1F). In the validation studies, GFP reactivation was L-type voltage-operated Ca2 channels (Supplementary Fig. S5A – tested at multiple doses (Fig. 2A F). All cardiac glycosides induced and S5B). Instead, YB5 cells have functional plasma membrane þ dose-dependent GFP reactivation in the nanomolar range at clin- store-operated Ca2 entry (SOCe) channels, which transport þ ically relevant concentrations, with proscillaridin A being the most extracellular Ca2 into the cytosol in response to a decrease in þ potent (Supplementary Fig. S2A and S2B). We also validated three Ca2 level in the endoplasmic reticulum (ER) store. The activity of additional antibiotics (pyrithion zinc, cycloheximide, and thiram) these plasma membrane channels was demonstrated by the effect fi þ in the antibiotic/others subgroup that were identi ed in the screen of SOCe inhibitors reducing cytosolic Ca2 entry triggered by the m þ at 50 mol/L only but were positive during the validation using ER Ca2 pump blocker, thapsigargin (Supplementary Fig. S5C; both schedules. Therefore, we validated a total of 14 FDA-approved refs. 17, 18). drugs encompassing three well-known epigenetic drugs and 11 Then, we characterized the effects of drug treatments in YB5 þ þ candidate epigenetic drugs. cells on cytosolic Ca2 levels at rest, ER Ca2 levels and SOCe þ activity in response to Ca2 ionophore, ionomycin or thapsigar- þ No changes in classical epigenetic pathways gin followed by the addition of 1 mmol/L Ca2 in the media (a We confirmed GFP mRNA reactivation after 24 hours treatment cation-safe solution). Interestingly, known epigenetic drugs did þ by qPCR (Fig. 3A). We also detected gene reactivation in three not alter Ca2 homeostasis (Fig. 4A). However, cardiac glycosides

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Figure 2. Validation of positive hits with known epigenetic drugs, cardiac glycosides, and antibiotics/others subgroups. Dose–response curves were generated measuring GFP expression in a validation step in YB5 cells treated for 24 hours (left) or 72 hours (right) with known epigenetic drugs (A and B), cardiac glycosides (C and D), and antibiotics/others (n 3; E and F).

þ þ and arsenic trioxide reduced ER Ca2 levels (first peak) while Ca2 levels in the ER (first peak; Fig. 4C). Thus, most of the þ þ strongly inhibiting SOCe-mediated Ca2 entry (second peak; Fig. candidate epigenetic drugs induce measurable intracellular Ca2 þ 4B and Supplementary Fig. S5D). Even though ER Ca2 levels changes in YB5 cells by different mechanisms of action, which þ were significantly reduced, we could not measure an increase in result in cytosolic Ca2 sparks or sustained increased levels. It is þ þ þ þ cytosolic Ca2 , suggesting a transient spark in cytosolic Ca2 after noteworthy that cardiac glycosides are well-known Na /K - treatment with digitalis compounds (19). In another model of ATPase pump blockers used in heart failure treatment (11). To þ þ HEK293 cells expressing the genetically encoded calcium sensors assess the impact of Na /K -ATPase pump inhibition on GFP (D1ER cameleon system) specifically expressed in the ER, we reactivation in YB5 cells, we treated YB5 cells with orthovanadate, þ þ þ confirmed digitoxin-induced reduction in ER Ca2 (Supplemen- an inorganic Na /K -ATPase inhibitor and did not detect GFP tary Fig. S5E; 20). Finally, we could detect an increased cytosolic reactivation. These data suggest that the epigenetic activity of þ þ þ þ Ca2 level (higher baseline Ca2 signal) after only pyrithion zinc cardiac glycosides might be independent of Na /K -ATPase (data treatment whereas both pyrithion zinc and disulfiram increased not shown).

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Figure 3. Gene reactivation by newly identified drugs is independent of DNA demethylation and chromatin modifications. A, GFP reactivation in YB5 cells treated for 24 hours, measured by qPCR (n ¼ 3). B, SFRP1, TSG is reactivated after treatment detected by GFP fluorescence in HCT116 SFRP1-GFP cell line. Cells were treated either 72 hours with decitabine (followed by 24 hours without drug) or 24 hours with the identified hits at doses indicated on the graph (n ¼ 3). C and D, CMV promoter (C) and LINE-1 (D) DNA methylation levels measured by bisulfite-pyrosequencing in YB5 cells treated with drugs and doses indicated on the graph (n ¼ 3). (Cyclohex., cycloheximide; Oxyquin., oxyquinoline; Prosc. A, proscillaridin A; Pyrith. Z., pyrithion zinc.), E, mass spectrometry showing histone acetylation levels at several residues in YB5 cells treated for either 72 hours or 24 hours with drugs and doses indicated on the graph (n ¼ 3).

We further explored the mechanism of SOCe inhibition not caused by changes in cytosolic pH (Supplementary Fig. induced by cardiac glycosides. SOCe is mediated by the highly S5H; refs. 20, 24, 25). Finally, digitoxin specifically inhibited þ þ Ca2 -selective Orai1 channels, which are activated by STIM pro- Ca2 influx through Orai1 in HEK293 cells expressing either þ teins in response to a decrease in ER Ca2 levels. STIM translocates constitutively active STIM1 mutant (STIM1D76A) or constitu- into ER-plasma membrane junctions to tether and activate Orai1 tively active Orai1 mutant (O1-V102C; Fig. 4E and F; 26). In channels (21, 22). For these experiments, we used a well-estab- these experiments, we used the antihistamine fexofenadine as a lished model of HEK293 cells overexpressing Orai1-CFP and control for specificity of action as this drug treatment does not STIM1-YFP (23). We found that digitoxin did not affect STIM1- induce GFP reactivation. Together, the data demonstrate that þ Orai1 coupling as maximal FRET signal between STIM1 and cardiac glycosides altered Ca2 levels by causing potent SOCe Orai1, obtained after ionomycin treatment, was unaltered by inhibition. digitoxin (Supplementary Fig. S5F). Wild-type and overexpressing þ STIM1-Orai1 HEK293 cells showed similar inhibition of SOCe Ca2 signaling through CamK is essential for gene reactivation þ and reduction in ER Ca2 size by digitoxin (Fig. 4D and Supple- CamK, a multifunctional serine/threonine kinase, is a central þ þ mentary Fig. S5G). Importantly, these effects on Ca2 levels were molecule that is activated by an increase in cytosolic Ca2 , leading

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Figure 4. Newly identiﬁed drugs induce changes in intracellular Ca2þ levels. Intracellular changes in Ca2þ levels in YB5 cells pretreated for 8 hours with known epigenetic drugs þ þ (A), cardiac glycosides (B), pyrithion zinc and disulﬁram (C) in response to ionomycin, extracellular Ca2 , and thapsigargin. D, time-lapse intracellular Ca2 þ changes in HEK293 cells overexpressing STIM1-Orai1 proteins pretreated for 8 hours with digitoxin in response to ionomycin and extracellular Ca2 . Time-lapse þ intracellular Ca2 changes in HEK293 cells transfected with constitutively active STIM1-mutant protein (D67A; E) or with constitutively active Orai-mutant channel (V102C; F) in response to extracellular Ca2þ exposure and SOCe inhibitor (2-APB). The arrows indicate the addition of Ca2þ into the extracellular media while a line indicates the time of Ca2þ exposure prior to its removal by washes of Ca2þ-free solution. Fexofenadine was used as a control as this drug does not induce GFP reactivation.

to gene expression changes in part caused by the unbinding of We next hypothesized that the epigenetic effects of CamK þ chromatin repressors such as the methyl-binding protein MeCP2 activation by Ca2 signaling relate to unbinding of MeCP2 from from their silenced promoters (15, 27–29). Except for known repressed promoters, as previously reported in normal neuronal epigenetic drugs, all candidate epigenetic drugs induce intracel- cells (15, 16, 28, 29). Using ChIP-qPCR following cardiac glyco- þ lular Ca2 changes. Thus, we tested the role of CamK in YB5 cells side treatment (Lanatoside C), we found that MeCP2 occupancy treated with known and candidate epigenetic drugs. Drug- was reduced at several DNA hypermethylated promoters (Sup- induced reactivation of GFP and endogenous TSGs (TIMP-3 and plementary Fig. S7), suggesting that drug-induced dissociation of WIF-1) was signiﬁcantly reduced by pharmacologic inhibition of MeCP2 binding is associated with gene reactivation. Most inter- CamKII using Kn-93 whereas Kn-92, its weaker analogue, had a estingly, using confocal microscopy, we observed nuclear versus much smaller effect (Fig. 5A and Supplementary Fig. S6A and S6B; cytoplasmic redistribution of MeCP2 after proscillaridin treat- ref. 30). As expected, GFP expression induced by azacitidine was ment (Fig. 5B). Following 24-hour and 48-hour treatment with not sensitive to Kn-93 inhibition as this hypomethylating drug proscillaridin at 500 nmol/L, MeCP2 staining was enriched in the þ did not alter Ca2 ﬂuxes (Fig. 5A). cytoplasm in most of the cells (Fig. 5B). In untreated cells, MeCP2

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Figure 5. Drug-induced gene reactivation is dependent on CamK activity. A, GFP reactivation by flow cytometry in YB5 cells pretreated (4 hours) with CamK inhibitor Kn-93 and its weaker analogue Kn-92 and treated for 24 hours. B, fluorescent immunochemistry in SW48 cells (YB5 parental cells) treated for 24 hours and 48 hours with proscillaridin at 500 nmol/L stained with DAPI, MeCP2 (RFP) antibody. White arrows, MeCP2 signal in the cytoplasm post-proscillaridin A treatment. C, percentage of apoptotic cells in YB5 cells pretreated (4 hours) with CamK inhibition and treated for 48 hours. staining was mainly concentrated in the nucleus and a weak signal Western blot experiments and confocal microscopy on YB5 was detected in the cytoplasm as already observed by others (31). untreated and treated with proscillaridin A) and HEK293T cells MeCP2 antibody specificity was verified by using siRNA (in overexpressing MeCP2 (Supplementary Figs. S8 and S9). Finally,

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þ we showed that these effects of the positive hits identified in the Ca2 signaling also occurred in our cancer cell screening system screen may relate to export of regulatory proteins from the nucleus (YB5 cells) and only after treatment with the newly epigenetic to the cytoplasm. Blocking the exportin pathway of protein drugs but not with well-known epigenetic drugs such as DNA transport from the nucleus to the cytoplasm by leptomycin methylation and HDAC inhibitors. This difference suggested a significantly reduced drug-induced GFP reactivation in YB5 cells separate mechanism of action. Our data showed that reactivation þ (Supplementary Fig. S10). of endogenous epigenetically silenced TSGs was linked to Ca2 As CamKII appeared to mediate gene reactivation, we next signaling and CamK activation. Mechanistically, our data suggest tested whether the selective cancer cell killing was also CamKII- that gene reactivation may involve gene derepression by inducing dependent. Percentages of apoptotic cells, viable cells, and dead MeCP2 eviction from repressed promoters to the cytoplasm cells were measured in colon cancer (YB5 and its parent cell line through the exportin pathway (Supplementary Fig. S14). Inter- SW48) and leukemia (K562 and HL-60) cell lines following 48- estingly, the anticancer activity could also be modulated by CamK hour drug treatment alone or in combination with CamKII pharmacologic inhibition. inhibitor, Kn-93 and its less active derivative, Kn-92. For all cell Consistent with our work, other studies have demonstrated a lines, drugs significantly induced cytotoxicity whereas, as expected CamK-dependent mechanism producing MeCP2 release from for this brief treatment period, hypomethylating drugs did not. silenced promoters, leading to gene reactivation (15, 28, 29). Kn-93 rescued almost completely the drug-induced toxic effects Interestingly, our data further demonstrate redistribution of (Fig. 5C and Supplementary Figs. S11–S13). These data suggest MeCP2 to the cytoplasm after drug-induced CamK activation. that CamK activation is a key effector of the anticancer activity of The relationship between MeCP2 binding to methylated-CpGs þ these FDA-approved drugs. and Ca2 has been described in neurons where MeCP2 phosphorylation is involved in intracellular localization during neu- Anticancer activity and cancer selectivity ronal differentiation (31, 32). Phosphorylation sites of MeCP2 are We next assessed the anticancer activity of the newly identified known to modify its binding to other epigenetic cofactors but candidate epigenetic drugs in YB5 cells. Anchorage-dependent more studies are needed to clearly understand the role of CamK colony formation assays were performed for long-term cytotox- and MeCP2 phosphorylation sites. Nevertheless, our study shows icity evaluation. All drugs reduced cancer cell survival in a dose that the CamK–MeCP2 interaction previously linked only to and time-dependent manner (Fig. 6A–C). Anticancer activity was differentiation can be exploited against cancer cells to reactivate stronger for the newly identified drugs compared with known silenced TSG. These data also demonstrate a key role for calcium epigenetic drugs. Anchorage-independent colony assays were fluxes in epigenetic silencing in neoplasia. then performed by gauging colony formation in soft agar. All Interestingly, we identified a class effect for the cardiac glycoside drugs produced a significant dose-dependent reduction in the subgroup where all these heart failure drugs present in the libraries number of colonies; with proscillaridin A being the most potent scored positive in the screen and also separately validated. These (Fig. 6D). To address the question of cancer cell selectivity, we drugs are relevant for cancer treatment because epidemiologic compared the viability of normal colon cells (CRL-1831) versus studies have shown that patients treated with cardiac glycosides colon cancer cells (SW48). We used YB5's parental cell line SW48 for heart failure exhibit a reduced cancer rate and less aggressive for this because GFP expression could affect viability measure- cancers (11, 33, 34). Moreover, in vitro studies have previously ments performed by flow cytometry. After 48-hour treatment at shown selective cancer killing for some of these cardiac glycosides, þ doses producing the strongest GFP reactivation in YB5 cells, cell which we now relate to Ca2 signaling and TSG reactivation (35). viability was reduced in cancer cells but not in normal cells (Fig. Our data suggest that their chemopreventive or chemotherapeutic þ 6E) demonstrating the cancer selectivity of these drugs. activity may be in part due to their ability to target Ca2 fluxes and reactivate epigenetically silenced TSGs. Another drug we identi- fi Discussion ed as having effects on gene silencing is arsenic trioxide, which has marked activity in acute promyelocytic leukemia (36), a There is a need to discover new epigenetic targets and drugs to disease characterized by a block in cell differentiation (37). Our induce TSG reactivation in cancer cells. The arsenal of epigenetic data suggest that epigenetic modulation may be part of its drugs approved in the clinic is currently limited to only four drugs antineoplastic mechanism of action. Interestingly, cytotoxic drugs targeting only two separate mechanisms. On the basis of the high or targeted drugs (e.g., tyrosine kinase inhibitors) used in cancer number of epigenetic targets in cancer, there is a great interest in chemotherapy did not reactivate GFP, pointing to the specificity of discovering new targets that can be used to reactivate TSGs and the identified hits. This was verified separately from the screen for reprogram cancer cells to eradicate their clonogenic potential and many cytotoxic drugs (38). Thus, the mechanism of action of the induce their differentiation. We used our live cell-based assay positive hits is likely to be specific disruption of epigenetic path- where we measured GFP reactivation as surrogate for TSG reac- ways involved in gene silencing rather than nonspecific effects tivation. Overall, we identified 14 hits including 3 well-known seen with many antiproliferatives. þ epigenetic drugs and 11 other FDA-approved drugs encompassing Finally, it is worth noting that Ca2 signaling is essential to cardiac glycosides and some antibiotics. initiating epigenetic reprogramming in early embryogenesis (39) To identify the mechanism responsible for GFP and endoge- and our data extend these findings to cancer therapy. Also, the nous TSGs reactivation, we first analyzed promoter DNA meth- mechanism of epigenetic action of these drugs is unlikely to be þ ylation and histone modifications. No changes were detected after traced to a single chromatin regulator; rather, Ca2 signaling treatment with the cardiac glycosides or the antibiotics while TSG through CamK activation may have effects on multiple epigenetic reactivation was detected. Surprisingly, we noticed that most of acting proteins simultaneously. It will be interesting to determine 2þ the newly identified drugs are known to induce alterations in Ca what targets, other than MeCP2, might be phosphorylated by signaling in normal cells. We demonstrated that these changes in CamK activation.

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Targeting Calcium Signaling Produces Epigenetic Reactivation

Figure 6. Anticancer efﬁcacy and selectivity of candidate epigenetic drugs. Anchorage-dependent toxicity assays were performed in YB5 cells treated for 72 hours (top) or 24 hours (bottom) with known epigenetic drugs (A), cardiac glycosides (B), and antibiotics/others (n ¼ 3; C). D, anchorage-independent clonogenic assays in soft agar were performed after a 24-hour treatment at the doses indicated on the graph (n ¼ 3). E, cell viability assays using normal colon cells (CRL-1831) and SW48 (YB5's parental cell line) after a 48-hour treatment, measured by ﬂow cytometry. Drugs and doses are indicated on the graph (n ¼ 3). Cyclohex, cycloheximide; Oxiquin, oxiquinoline; pyrithion, Pyrition zinc.

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Raynal et al.

þ In summary, our data identify Ca2 signaling as a new pathway Writing, review, and/or revision of the manuscript: N.J.-M. Raynal, J.T. Lee, that can be targeted to reactivate TSGs in cancer. This study suggest Y. Wang, J. Garriga, S. Dumont, W.E. Childers, R.A. Henry, J. Jelinek, S.B. Baylin, that cardiac glycosides and other FDA-approved drugs such as D.L. Gill, J.-P.J. Issa Administrative, technical, or material support (i.e., reporting or organizing some antibiotics represent interesting drugs for cancer epigenetic data, constructing databases): N.J.-M. Raynal, J.T. Lee, W. Chung, Y. Cui chemotherapy aiming to reactivate epigenetically silenced TSGs. Study supervision: S.B. Baylin, J.-P.J. Issa Other (perform experiments): S. Ahmed Disclosure of Potential Conﬂicts of Interest Other (assisted with selection of compounds tested in this study): No potential conﬂicts of interest were disclosed. W.E. Childers Other (collaboration by applying HTS using the Center's screening capabil- Authors' Contributions ities and compound library): M. Abou-Gharbia Conception and design: N.J.-M. Raynal, Y. Wang, S.B. Baylin, D.L. Gill, J.-P.J. Issa Grant Support Development of methodology: N.J.-M. Raynal, Y. Wang, P. Madireddi, J.-P.J. Issa is an American Cancer Society Clinical Research professor sup- J. Garriga, G.G. Malouf, E.J. Dettman, V. Gharibyan, W. Chung, M. Abou- ported by a generous gift from the F.M. Kirby Foundation. This work was Gharbia, D.L. Gill supported by NIH grants CA100632 and CA046939 (J.-P.J. Issa). Acquisition of data (provided animals, acquired and managed patients, The costs of publication of this article were defrayed in part by the provided facilities, etc.): N.J.-M. Raynal, J.T. Lee, Y. Wang, A. Beaudry, G. payment of page charges. This article must therefore be hereby marked Malouf, S. Dumont, E.J. Dettman, R.A. Henry, S.B. Baylin advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Analysis and interpretation of data (e.g., statistical analysis, biostatistics, this fact. computational analysis): N.J.-M. Raynal, Y. Wang, G.G. Malouf, S. Dumont, E.J. Dettman, V. Gharibyan, W. Chung, W.E. Childers, R.A. Henry, A.J. Andrews, Received August 13, 2014; revised December 3, 2015; accepted December 18, J. Jelinek, S.B. Baylin, D.L. Gill, J.-P.J. Issa 2015; published OnlineFirst December 30, 2015.

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Targeting Calcium Signaling Induces Epigenetic Reactivation of Tumor Suppressor Genes in Cancer

Noël J.-M. Raynal, Justin T. Lee, Youjun Wang, et al.

Cancer Res 2016;76:1494-1505. Published OnlineFirst December 30, 2015.

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