Byun et al. Cell Death and Disease (2019) 10:689 https://doi.org/10.1038/s41419-019-1892-7 Cell Death & Disease

ARTICLE Open Access Epigenetic re-wiring of by pharmacological targeting of C-terminal binding Jung S. Byun1,SamsonPark2,DaeIkYi2, Jee-Hye Shin3,SaraGilHernandez2, Stephen M. Hewitt4,MarcC.Nicklaus5, Megan L. Peach6, Laura Guasch5,BinwuTang3, Lalage M. Wakefield3,TingfenYan7,AmbarCaban1, Alana Jones 1, Mohamed Kabbout1, Nasreen Vohra8, Anna María Nápoles1, Sandeep Singhal9, Ryan Yancey9,AdrianaDeSiervi10 and Kevin Gardner1,9

Abstract The C-terminal binding protein (CtBP) is an NADH-dependent dimeric family of nuclear that scaffold interactions between transcriptional regulators and chromatin-modifying complexes. Its association with poor survival in several cancers implicates CtBP as a promising target for pharmacological intervention. We employed computer- assisted drug design to search for CtBP inhibitors, using quantitative structure-activity relationship (QSAR) modeling and docking. Functional screening of these drugs identified 4 compounds with low toxicity and high water solubility. Micro molar concentrations of these CtBP inhibitors produces significant de-repression of epigenetically silenced pro- epithelial , preferentially in the triple-negative breast cancer cell line MDA-MB-231. This epigenetic reprogramming occurs through eviction of CtBP from promoters; disrupted recruitment of chromatin-modifying protein complexes containing LSD1, and HDAC1; and re-wiring of activating marks at targeted genes. In

1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; functional assays, CtBP inhibition disrupts CtBP dimerization, decreases cell migration, abolishes cellular invasion, and improves DNA repair. Combinatorial use of CtBP inhibitors with the LSD1 inhibitor pargyline has synergistic influence. Finally, integrated correlation of in breast cancer patients with nuclear levels of CtBP1 and LSD1, reveals new potential therapeutic vulnerabilities. These findings implicate a broad role for this class of compounds in strategies for epigenetically targeted therapeutic intervention.

Introduction recruiting a diverse array of chromatin-modifying com- The C-terminal binding protein (CtBP) was first plexes2,4. Binding partners for CtBP include histone dea- described as a phosphoprotein that binds specifically to cetylases, histone methyltransferases, and histone – the C-terminal end of the E1a adenovirus oncogene1 3. in addition to several different classes of These proteins were later found to represent a dimeric sequence-specific DNA binding proteins and chromatin- family of proteins, composed of CtBP1 and CtBP2, that associated complexes2,3. Therefore, in its dimeric form, can homodimerize or heterodimerize in the nucleus to CtBP has the broad potential of re-shaping the landscape of influence multiple different epigenetic nuclear events by epigenetic regulation throughout the nucleus5,6.CtBP belongs to a family of NAD-dependent D-2-hydroxy acid dehydrogenases including E. Coli D-3-phosphoglycerate Correspondence: Kevin Gardner ([email protected]) 1National Institute on Minority Health and Health Disparities, Bethesda, MD dehydrogenase, bacterial D-lactate dehydrogenase (D- 20892, USA LDH) and D-hydroxyisocaproate dehydrogenase7.Though 2 – Genetics Branch, National Cancer Institute, Bethesda, MD 20892, USA the actual substrate for CtBP remains unclear8 10, its ability Full list of author information is available at the end of the article. These authors contributed equally: Samson Park, Dae Ik Yi to dimerize and form higher order oligomers is positively Edited by B. Zhivotovsky

© The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Official journal of the Cell Death Differentiation Association Byun et al. Cell Death and Disease (2019) 10:689 Page 2 of 15

regulated by NADH/NAD+7,11. The ability of CtBP to identified are shown in Fig. 1b. These 24 compounds were bind and undergo redox cycles with NADH/NAD+ and then experimentally screened for influence on viability substrate implicates a substantial role for CtBP in the and proliferation by MTT assay (Fig. 1c) and combined regulation of genomic responses to changes in cellular viability, cytotoxicity and apoptosis assay (Fig. 1d). metabolism9,12. CtBP functions as both an activator and repressor of CtBP levels are elevated in multiple different cancers to transcription although most interactions described thus profoundly influence cellular phenotypic plasticity by far are repressive2. The transcriptionally repressive activ- promoting pathways linked to epithelial-to-mesenchymal ity of CtBP is ascribed to its ability to bind and recruit a transition, cell migration, decreased genome stability and variety of chromatin-modifying enzymes that remove – the acquisition of stem cell self-renewal features13 16. The activating chromatin modifications including histone 3 increasing role of epigenetic regulation in tumor hetero- and 4 acetylation and trimethylation of histone 3 at geneity, cellular plasticity and the acquisition of drug lysine 42,3. However, the function of CtBP is highly cell- resistance17 suggests a significant potential function for type specific, in part due to the differential expression of CtBP as a major determinant in the epigenetic control of various chromatin regulatory complexes and the differ- cancer. These dramatic properties implicate CtBP as a ential abundance of proteins that destabilize and, there- promising candidate for targeted disruption by small fore, downregulate CtBP activity including: APC, HIPK2, molecule inhibitors as a therapeutic approach against AMPK, and JNK12,3. In breast cancer, over-expression of – cancer18 23. The first proof of this principle was provided CtBP is associated with downregulation of a variety of by the discovery that 2-Keto-4-methylthiobutyrate pro-epithelial genes in ER+ tumors but has little effect on (MTOB), an intermediate in methionine metabolism, is the expression of these genes in breast cancer cell types a selective inhibitor of CtBP activity capable of disrupting with more mesenchymal or stromal features14. Down- tumor growth in murine models10,18. However, MTOB regulation or depletion of CtBP by RNAi-mediated gene requires 10 mM concentration to be effective and is depletion results in upregulation of pro-epithelial genes in therefore considered impractical as a therapeutic agent10. the mesenchymal breast cancer cell line MDA-MB-23114. Recently, the crystallographic structure of the dehy- We applied a functional screen for the loss of CtBP drogenase domains of both CtBP2 and CtBP1 in complex transcriptional repressive activity by screening the 24 with MTOB and NAD+ has been resolved20. This compounds for the ability to upregulate FOXA1 and advance provided a framework through which more OVOL2, two genes that are intimately involved in main- – effective CtBP inhibitors were designed through compu- taining the epithelial phenotype25 28. This functional – tational methods20 22. Using a similar approach, 24 screen identified several compounds that substantially commercially available compounds with potential as CtBP (greater than two-fold) upregulate FOXA1 and/or OVOL2 inhibitors were identified. Four lead compounds were expression (Fig. 1e). From this screen, 4 compounds selected from these candidates based on their solubility, (CI19, CI22, CI23, and CI24) showing both the lowest low cytotoxicity and ability to reverse transcriptional cytotoxicity, and upregulated FOXA1 and OVOL2 repression by CtBP. Further characterization of these expression in MDA-MB-231, were selected (Fig. 1e). All compounds indicates that they have potent activity four of these compounds have molecular weights less than against CtBP at low micromolar concentrations to induce 300 Da and are water-soluble (Fig. 1f). significant alterations in epigenetic transcriptional pro- gramming in breast cancer. CtBP inhibitors disrupt CtBP dimerization in vivo CtBP activity is dependent on its ability to form dimers Results and higher order oligomers. This property enables CtBP Identification of small molecular inhibitors of CtBP to recruit chromatin-modifying complexes to specific We exploited the observation that most dehydrogenases chromatin locations and stabilizes CtBP against nuclear have strict substrate specificities and the recent publica- export and degradation2,3. As shown in Fig. 2, assessment tion of the crystallographic structure of MTOB in com- of CtBP1/CtBP2 dimerization by fluorescent resonance plex with CtBP20 to conduct virtual screening of the energy transfer (FRET) acceptor photobleaching29 reveals ChemNavigator iResearch Library from Sigma Aldrich24 a significant decrease in the paired CtBP1-YFP/CtBP2- to select molecules that showed favorable interactions CFP FRET signal, after incubation with CI19, CI22, CI23 with three residues (His315, Glu295, Arg 266) demon- or CI24 (Fig. 2a, b). This is consistent with the observation strated to function as a catalytic triad in the active site of that CI19, CI22, CI23, and CI24 are also able to induce the CtBP8. This computational screen identified 31 com- release of CtBP2 from immuno-precipitated CtBP1 pounds of which 24 were commercially available. The complexes in vitro (Supplementary Fig. 2). Finally, the docked structures of four representative compounds are dose-response for CI24 suggests the IC50 for CtBP shown in Fig. 1a and the structures of the 24 compounds dimerization lies between 10 and 20 μM (Fig. 2c).

Official journal of the Cell Death Differentiation Association Byun et al. Cell Death and Disease (2019) 10:689 Page 3 of 15

Fig. 1 Identification and validation of small molecule CtBP inhibitors by computer-assisted drug design using QSAR-based modeling. a Representative docked structures of four small molecular inhibitors in the active site of CtBP. Four lead compounds (CI19, CI22, CI23, and CI24) are shown in green in the CtBP substrate binding site. The NAD+ cofactor is colored in light blue. Hydrogen bonds are indicated with dashed black lines. b Structures of 24 commercially available predicted inhibitors of CtBP screened based on best QSAR predicted activities and highest docking scores. c Proliferation and viability screening of MDA-MB-231 cells treated 24 h with 10 μM of the 24 predicted CtBP inhibitors as determined by MTT assay. d Viability, cytotoxicity, and apoptosis screen of MDA-MB-231 cells treated 24 h with 10 μM of the 24 predicted CtBP inhibitors as assessed by the ApoTox-Glo™ Triplex Assay. Samples were normalized and subject to hierarchical clustering using a correlation matrix and centroid linkage. Dendrogram highlighted in red contains the 4 lead compounds with the lowest cytotoxicity and highest de-repression of CtBP target genes. e qRT- PCR profiling of mRNA abundance of CtBP-repressed genes, FOXA1 and OVOL2, in MDA-MB-231 cells after treatment for 24 h with 10 μM of the indicated CtBP inhibitors. f Table showing the relative water solubility of the 4 lead compounds based on AlogP (octanol/water partition coefficient) and QPlogS (predicted aqueous solubility). All experiments represent the average and standard error of at least two biological replicates. The error bars represent the s.d. of the mean from at least two independent experiments.*Indicates p-values < 0.05 and **indicated p-value < 0.001

Disruption of epigenetic control of transcription by CtBP CtBP chromatin immunoprecipitation, using an antibody small-molecule inhibition that recognizes both CtBP1 and CtBP213,14, demonstrates These four lead compounds were then screened for that treatment of MDA-MB-231 cells with 10 μM of the their specific ability to target and de-repress pro-epithelial CtBP inhibitor results in eviction of CtBP from the pro- genes that are known to be transcriptionally silenced in moter regions of the master epithelial regulatory genes the mesenchymal triple negative breast cancer cell line OVOL2, GATA3, FOXA1, and GRHL214, in addition to the MDA-MB-23130,31. As shown in Fig. 3a, b, analysis by pro-epithelial micro-RNAs, miR200c and miR203. This

Official journal of the Cell Death Differentiation Association Byun et al. Cell Death and Disease (2019) 10:689 Page 4 of 15

CtBP Dimerization A (CtBP1-YFP/CtBP2-CFP) B C

pre-photobleaching 100

1.0 Signal ** CI24 ** 50 24 h post-photobleaching 0.5 ** **

0.0

% Maximum FRET Signal % Maximum FRET 0

% Maximum FRET 0 102030405060708090100 eh V 19 22 23 24 CI24 [uM]

Fig. 2 Pharmacological Inhibition of CtBP1/CtBP2 heterodimer formation by CtBP inhibitors. a CtBP1-YFP and CtBP2-CFP heterodimerization was measured by fluorescence resonance energy transfer (FRET) acceptor photobleaching (see Materials and methods). b Inhibition of CtBP dimerization FRET signal by the CtBP inhibitors CI19, CI22, CI23, and CI24 at 10 μM concentrations. c Dose-response curve of inhibition of CtBP dimerization FRET signal by increased concentrations of CI24 CtBP inhibitor. The error bars represent the s.d. of the mean from at least two independent experiments. *Indicates P < 0.05 and **indicates P < 0.01. Error bars in c are smaller than the size of the symbols

CtBP promoter eviction is associated with significant have been commonly shown to associate with CtBP are upregulation of the RNAs for OVOL2, GATA3, FOXA1, the histone deacetylases HDAC1 and HDAC2 and the GRHL2,miRNAmiR200c, miR203, and the well-known H3K4Me2/1 , LSD16,34,35. LSD1 (KDM1A)is CtBP-repressed genes, E-Cadherin and BRCA113,32 (Fig. 3c). a that catalyzes the removal of This gene reactivation in MDA-MB-231 is reflected by an H3K4Me2 and K3K4Me1 activation marks by demethy- increased level of protein expression (Fig. 3d, bottom). lation, thus disrupting the ultimate accumulation of the Notably, as shown in Fig. 3d, this drug-induced eviction active H3K4Me3 modification to play a pivotal role in occurs in the absence of appreciable loss or shift in neither modulating epithelial-to-mesenchymal transition36.By total CtBP1 nor CtBP2 protein levels from the cytosolic and ChIP analysis, the addition of the CtBP inhibitors results nuclear compartments (Fig. 3d, top). The relatively modest in significant eviction of HDAC1 from the OVOL2, increase in both OVOL2 and FOXA1 protein levels likely FOXA1, GRHL2, GATA3, miRNA 200c, and miRNA 203 reflects differential influences of both post-transcriptional promoters (Fig. 5a) with a similar pattern for LSD1 ChIP (RNA stability and decay) and post-translational steps in (Fig. 5b). Accordingly, loss of LSD1 and HDAC1 from OVOL2 and FOXA1 regulation. these respective promoter regions is associated with sig- A survey comparing the CtBP inhibitor-induced tran- nificant increases in both histone 4 acetylation and the scriptional de-repression of the pro-epithelial genes in cell deposition of H3K4Me3 marks at the promoter regions of lines characterized by higher levels of epithelial differ- GATA3, GRHL2, FOXA1, OVOL2, miRNA 200c, and entiation, including the estrogen- positive MCF-7 miRNA 203 (Fig. 5c). cell line, and the non-transformed mammary epithelial cell line MCF-10A is shown in Fig. 4. For each compound, CtBP inhibition increases DNA repair and blocks cell this survey demonstrates similar trends but lower levels of migration and invasion transcriptional reactivation by the CtBP inhibitor com- One of the most characterized influences of CtBP on pounds in both MCF-7 and MCF-10A (Fig. 4). These cellular phenotype is its role in promoting cellular findings indicate a cell-specific dose-sensitivity to CtBP migration, a key gain of function in cells undergoing inhibition. epithelial-to-mesenchymal transition and a central feature during tumor metastasis32. As shown in Fig. 6a, RNAi- CtBP inhibitors disrupt recruitment of histone modification mediated depletion of CtBP in MDA-MB-231 leads to a machinery demonstrable decrease in cell migration, compared to Multiple studies have shown that CtBP ferries different empty vector control (pGIPz) as measured by a wound chromatin-modifying complexes to chromatin depending closure assay (right). Incubation of wild type MDA-MB- – on the promoter or enhancer context13 15,33. These 231 cells with 10 μM CtBP inhibitors decreases cell recruited assemblies then affect changes in the local epi- migration throughout the time course of the wound clo- genetic marks specific to the composition of the assem- sure assay with greater influences caused by compounds bled complexes. Two classes of chromatin modifiers that CI23 and CI24 (Fig. 6a, right). Similarly, at 10 micromolar

Official journal of the Cell Death Differentiation Association Byun et al. Cell Death and Disease (2019) 10:689 Page 5 of 15

A chr8: 130 _ 1 kb chr14: 23 _ 2 kb

1 _ 1 _ >>

GRHL2 >>>> >> FOXA chr10: 14 _ 2 kb chr20: 2 kb 31 _

1 _ 1 _

GATA3 >> > OVOL2 chr14: 18 _ 1 kb chr12: 1 kb 6.32 _

1 _ 1 _ MIR203 MIR200c B 2.5 5 OVOL2 CtBP GATA3 CtBP FOXA1 CtBP

2.0 Upstream 4 Upstream Promoter Promoter D 1.5 3 *

Relative Enrichment 1.0 2 * * * * Relative Enrichment ** * M Normalized to Input

Normalized to Input * r * Normalized to Input * 0.5 ** 1 * Veh 19 22 23 24 CtBP ChIP CtBP CtBP ChIP Relative Enrichment ChIP CtBP 0.0 ChIP CtBP 0 CtBP1 9 3 4 9 2 3 4 2 1 2 2 2 I1 I2 I veh I I I I (cytoplasm) veh C CI 22 C C veh C C C C 47- 5 5 5 GRHL2 CtBP miRNA 203 CtBP miRNA 200 CtBP CtBP2 4 4 Upstream (cytoplasm) Upstream 4 Upstream 47- Promoter Promoter Promoter 3 3 3 GAPDH * * * 34- (cytoplasm) 2 * 2 Relative Enrichment 2 * * * * * ** Relative Enrichment CtBP1 Normalized to Input ChIP Normalized to Input 1 Normalized to Input 1 1 ChIP (nuclear) * * CtBP CtBP 0 Relative Enrichment ChIP CtBP 0 0 CtBP2 9 2 4 9 4 9 2 3 4 2 1 22 1 2 2 2 I I2 I I I23 I2 veh I I I I (nuclear) veh CI 1 C CI 23 C veh C C C C C C C C GAPDH BRCA1 mRNA FOXA1 mRNA CDH1 mRNA miRNA 200c (nuclear) C 3 4 4 ** ** 4 ** ** ** ** ** ** M 3 r Veh 19 22 23 24 3 3 ** 2 ** ** ** ** ** ** 210- -PCR BRCA1 2 2 2 ** 101- 1 GRHL2 1 1 1 56.2-

mRNA qRT mRNA FOXA1 0 0 0 0 56.2- (normalized to 18s RNA) h 9 2 3 4 9 2 3 4 9 2 3 4 h 9 3 4 2 2 1 2 2 2 eh 1 2 2 1 22 2 2 Ve I1 I2 I I Veh I I I I V I I I Ve I I I GATA3 C C C C C C C C C C CI 2 C C C CI C OVOL2 mRNA GATA3 mRNA GRHL2 mRNA miRNA 203 125- CDH1 5 ** 3 4 ** ** 6 ** 29- OVOL2 4 ** ** ** ** ** 3 **

-PCR ** 2 ** ** T 3 4 35.8- GAPDH 2 ** 2 (whole cell) 1 * 1 2 ** * 1 mRNA qR mRNA

(normalized to 18s RNA) 0 0 0 0 2 4 h 2 2 4 h 9 2 3 4 h 2 23 2 e 23 24 19 2 23 2 e 1 2 2 Ve I19I I I V I19I2 I Veh I I I I V I I I C C C C C C C CI C C C C C C C CI 2

Fig. 3 Epigenetic reactivation of pro-epithelial gene expression in MDA-MB-231 cells following treatment with CtBP inhibitors. a Diagram of CtBP ChIP-seq profile14 used to select amplicon regions for detecting CtBP occupancy at the proximal promoter and upstream regions of the GRHL2, FOXA1, GATA3, OVOL2, miRNA 203, and miRNA 200c genes. b qChIP profile of CtBP binding to the promoter and upstream regions of OVOL2, GATA3, FOXA1, GRHL2, miRNA 203, and miRNA 200c following treatment of MDA-MB-231 cells with the vehicle and 10 μM CI19, CI22, CI23, and CI24 inhibitors for 24 h prior to ChIP. c qRT-PCR profiles of BRCA1, FOXA1, CDH1, OVOL2, GATA3, and GRHL2 mRNA and miRNA 200c and miRNA 203 abundance in MDA-MB-231 cells treated 24 h with 10 μM CtBP inhibitors as indicated. d (Top), immuno-blot profiling of CtBP1 and CtBP2 in nuclear and cytoplasmic extracts of MDA-MB-231 treated 24 h with vehicle or 10 μM of the indicated CtBP inhibitors. (Bottom), immuno-blot profiling of BRCA1, GRHL2, FOXA1, GATA3, CDH1, OVOL2, and GAPDH protein expression in whole-cell lysates of MDA-MB-231 cells, treated as described above. The error bars represent the s.d. of the mean from at least two independent experiments. P-values are calculated from the Students to T-Test relative to the vehicle control. *Indicates P < 0.05 and **indicates P < 0.01

Official journal of the Cell Death Differentiation Association Byun et al. Cell Death and Disease (2019) 10:689 Page 6 of 15

4.0 3.5 ** 3.0 FOXA1 3.0 GATA3 4.5 OVOL2 4.0 ** 2.5 2.5 ** ** 3.5 ** 2.0 ** 2.0 ** ** 3.0 1.5 ** ** 2.5 1.5 2.0

mRNA qRT-PCR mRNA ** 1.0 1.0 1.5 ** **

(normalized to 18s RNA) 0.5 1.0 0.5 0.5 0.0 0.0 0.0

eh eh eh eh eh

MCF7-V MCF7-V MCF7-CI24MCF7-CI23 MCF7-CI24MCF7-CI23 MCF7-V MDA-231-V MCF10A-Veh MCF10A-Veh MCF7-CI24MCF7-CI23 MDA-231-CI24MDA-231-CI23 MCF10A-CI24MCF10A-CI23 MDA-231-VMDA-231-CI23 MCF10A-CI24MCF10A-CI23 MDA-231-Veh MCF10A-Veh MDA-231-CI24 MDA-231-CI24MDA-231-CI23 MCF10A-CI24MCF10A-CI23

3.0 4.0 ** 4.0 ** ** 3.5 CDH1 3.5 GRHL2 2.5 BRCA1 3.0 3.0 ** ** 2.0 ** 2.5 2.5 -PCR 1.5 ** 2.0 2.0 ** 1.5 **** ** 1.5 ** 1.0 1.0 1.0

mRNA qRT mRNA 0.5 0.5 0.5

(normalized to 18s RNA) 0.0 0.0 0.0 eh eh eh eh eh eh

MCF7-V MCF7-V MCF7-CI24MCF7-CI23 MCF7-CI24MCF7-CI23 MCF10A-Veh MCF7-V MCF7-CI23 MCF10A-V MDA-231-Veh MCF10A-CI24MCF10A-CI23 MCF7-CI24 MDA-231-VehMDA-231-CI23 MCF10A-CI24MCF10A-CI23 MDA-231-CI24MDA-231-CI23 MDA-231-V MCF10A-V MDA-231-CI24 MDA-231-CI24MDA-231-CI23 MCF10A-CI24MCF10A-CI23

Fig. 4 The magnitude of de-repression of pro-epithelial genes by CtBP inhibitors is cell-type specific. qRT-PCR profiles of FOXA1, GATA3, OVOL2, CDH1, GRHL2, and BRCA1 mRNA in the presence of the 10 μM CtBP inhibitors CI23, and CI24, in MDA-MB-231, MCF-7, and MCF10A cell lines. The vehicle (Veh) used for all cell lines was phosphate-buffered saline. Results show the average and standard error of the mean (SEM) for three biological replicates. P-values are calculated from the Student’s T-Test relative to the vehicle control. *Indicates P < 0.05 and **indicates P < 0.01 concentration, all 4 compounds inhibit both invasion and used in the past alone or in combination with HDAC migration as demonstrated in Matrigel® invasion chamber inhibitors to induce growth inhibition and apoptosis in assays (Fig. 6b). TNBC cells39,42,44. Notably, the combination of these CtBP forms complexes at the promoters of numerous drugs were found to be more than additive for TNBCs genes involved in DNA repair, including the BRCA1 and additive or competitive in non-TNBC cell lines39. promoter and numerous members of the Fanconi Anemia Because CtBP is a well-characterized HDAC associated complementation group5,13,14,23. Prior studies have shown component of chromatin-modifying complexes, we that depletion of CtBP results in increased DNA repair explored the effectiveness of combining LSD1 inhibition detectable in both comet and gamma H2A.X foci for- with CtBP inhibition as a surrogate for HDAC disruption. mation assays14,37. In gamma H2A.X-labeled DNA repair As shown in Fig. 7a, while the addition of pargyline alone foci assays, the addition of either CtBP inhibitors CI19, minimally reactivates pro-epithelial gene expression in CI22, CI23, or CI24 at 10 micromolar concentrations MDA-MB-231, its combination with CtBP inhibitors causes increases in DNA repair as demonstrated by the show additive to synergistic responses in the de- significant decrease in remaining repair foci 24 h follow- repression of pro-epithelial genes, particularly with ing exposure to ionizing radiation (Fig. 6c, d). Notably, respect to CI24. A comparison of the functional influ- the increase is comparable and, in some cases, exceeds ences of pargyline and CI24 alone and in combination on that caused by RNAi-mediated CtBP gene depletion cellular migration was profiled over time (24 h) using the (Fig. 6d)14. IncuCyte ZOOM™ assay. As shown in Fig. 7b, while both CI24 and pargyline repress migration in this assay, their Synergistic pharmacological targeting of LSD1 in combination produces greater than additive effects. combination with CtBP Logistic regression reveals that the combination of CI24 LSD1 was one of the first chromatin regulatory com- and pargyline have their greatest influence over time in plexes to be found in association with CtBP6. Several the migration assay (Fig. 7c, d). Using the T-statistic, the recent studies have suggested the value of targeting LSD1 combination of Pargyline and CI24 have significantly in therapeutic strategies to epigenetically treat breast greater than additive influence on TNBC cell migration: cancer alone or in combination with other epigenetic (p-value = 5.27 E−08) compared to CI24 (p-value = – disruptors38 43. The LSD1 inhibitor, pargyline has been 0.249) or Pargyline (p-value = 0.0002) alone.

Official journal of the Cell Death Differentiation Association Byun et al. Cell Death and Disease (2019) 10:689 Page 7 of 15

A B 6 LSD1 GRHL2 8 LSD1 FOXA1 6 HDAC1 GRHL2 8 HDAC1 FOXA1 Upstream Upstream 6 Promoter Upstream 4 Promoter Upstream Promoter 6 Promoter 4 * * 4 ** * * * 4 2 * 2 2 * Relative Enrichment * ** Normalized to Input * * 2 ** Normalized to Input Normalized to Input 0 Normalized to Input ** ** ** 0

LSD1 ChIP Relative Enrichment LSD1 ChIP ris 0 0 Tris Relative Enrichment LSD1 ChIP T CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24

ris HDAC1 ChIP ris HDAC1 ChIP Relative Enrichment HDAC1 ChIP T T CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24 3 5 5 HDAC1 OVOL2 6 HDAC1 GATA3 LSD1 OVOL2 LSD1 GATA3 Upstream Upstream Upstream 4 Upstream 4 Promoter Promoter Promoter Promoter 2 4 3 3 * * 2 * * * * 2 * 2 1 **

* Relative Enrichment ** ** 1 ** ** 1 ** Normalized to Input ** ** Normalized to Input Normalized to Input Normalized to Input 0 0 0 0 LSD1 ChIP Relative Enrichment LSD1 ChIP HDAC1 ChIP Relative Enrichment HDAC1 ChIP

LSD1 ChIP ris Tris Tris T HDAC1 ChIP Relative Enrichment HDAC1 ChIP Tris CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24 6 15 5 8 HDAC1 miR200c HDAC1 miR203 LSD1 miR200c LSD1 miR203 4 Upstream Upstream Upstream 6 Upstream Promoter Promoter Promoter 4 Promoter 10 3 * * * 4

2 Relative Enrichment Relative Enrichment 5 ** Relative Enrichment 2 * ** 2 ** * ** ** ** Normalized to Input * 1 Normalized to Input

Normalized to Input ** ** ** Normalized to Input ** 0 0 LSD1 ChIP

0 0 LSD1 ChIP

HDAC1 ChIP Tris Tris HDAC1 ChIP Relative Enrichment HDAC1 ChIP ris ris T T CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24

C GATA3 H4Ac GRHL2 H4Ac GATA3 H3K4Me3 GRHL2 H3K4Me3 Upstream 20 15 Upstream 5 4 Promoter * Upstream Upstream * Promoter Promoter * Promoter * 4 15 * 3 10 * Relative Relative * * 3 * 10 2 2 Relative Enrichment 5 5 1 Normalized to Input Normalized to Input 1 H3K4Me3 ChIP H3K4Me3 ChIP H4Ac ChIP H4Ac ChIP Relative Enrichment H4Ac ChIP 0 0 0 0 Enrichment Normalized to Input Enrichment Normalized to Input ris ris ris T T Tris T CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24

25 FOXA1 H4Ac 10 OVOL2 H4Ac 15 FOXA1 H3K4Me3 10 OVOL2 H3K4Me3 Upstream Upstream * Upstream * Upstream Promoter Promoter Promoter 20 * * Promoter * 8 ** * 10 ** ** 15 * * * 6 * 5 ** 10 * 4 Relative Enrichment 5 Normalized to Input Normalized to Input 5 2 H3K4Me3 ChIP Relative H3K4Me3 ChIP H3K4Me3 ChIP Relative H3K4Me3 ChIP H4Ac ChIP H4Ac ChIP Relative Enrichment H4Ac ChIP 0 0 0 0 Enrichment Normalized to Input Enrichment Normalized to Input ris ris Tris Tris T T CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24 5 6 8 6 miR200c H4Ac miR 203 H4Ac ** miR200c H3K4Me3 miR203 H3K4Me3 Upstream Upstream 4 * Upstream ** 6 Upstream ** Promoter Promoter Promoter * 4 Promoter * 4 * ** ** Relative 3 * * * 4 * * ** 2 2 2 2 Normalized to Input Normalized to Input 1 H3K4Me3 ChIP Relative H3K4Me3 ChIP H3K4Me3 ChIP Enrichment Normalized to Input H4Ac ChIP Relative Enrichment H4Ac ChIP Enrichment Normalized to Input H4Ac ChIP Relative Enrichment H4Ac ChIP 0 0 0 0 ris T Tris Tris Tris CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24 CI19 CI22 CI23 CI24

Fig. 5 (See legend on next page.)

Official journal of the Cell Death Differentiation Association Byun et al. Cell Death and Disease (2019) 10:689 Page 8 of 15

(see figure on previous page) Fig. 5 Treatment with CtBP inhibitors is associated with LSD1 and HDAC1 eviction and increased deposition of activating chromatin marks at the promoter-proximal regions of pro-epithelial genes in MDA-MB-231. a qChIP profiles in MDA-MB-231 cells of HDAC1 eviction from the GRHL2, FOXA1, OVOL2, GATA3, miRNA 200c, and miRNA 203 proximal promoter regions following 24-h incubation with vehicle or 10 μM CtBP inhibitors. b qChIP profiles of LSD1 eviction from the GRHL2, FOXA1, OVOL2, GATA3, miRNA 200c, and miRNA 203 proximal promoter regions following 24-h incubation with vehicle or 10 μM CtBP inhibitors. c qChIP profiles of changes in histone 4 (K5,8,12,16) pan-acetylation (left) and histone 3 lysine 4 trimethylation (right) at the promoters and upstream regions of GATA3, GRHL2, FOXA1, OVOL2, miRNA 203, and miRNA 200c following 24-h incubation of MDA-MB-231 cells with 10 μM CtBP inhibitors. The error bars represent the s.d. of the mean from at least two independent experiments. P-values are calculated from the Student’s T-Test relative to the vehicle control. *Indicates P < 0.05 and **indicates P < 0.01

A Migration Assay C CtBP shCtBP1/2 Veh shRNA CI 19 CI 22 CI 23 CI 24 15 100 Veh pGIPZ ctrl CI 19 CI 22 0h 80 CI 23 6 hour CI 24 30 * 10 * 60 ** 20 12h ** 40 5 10 20

Wound Closure % Wound 24h

Wound Closure % Wound 0 Wound Closure % Wound 0 0 gamma H2Ax 9h 12h 0 5 10 15 20 25 30 Hours BD Veh Veh CI 19 30 CI 19 CI 23 CI 22 shCtBP CI 22 CI 24 CI 23 100 Veh Veh * CI 24 ** 20 ** ** ** ** ** ** ** ** ** , % Pos Nuclei 50 CI19 CI22

(cell per hpf) 10 ** ** ** ** ** 0 H2AX Foci

γ hours Transwell Migration/Invasion Transwell 0 0 0 2 4 0 4 0 2 4 0 2 4 0 2 4 CI23 CI24 12 24 1 2 12 2 1 2 1 2 1 2 Veh CI 19 CI 22CI 23 CI 24

Fig. 6 Pharmacologic inhibition of CtBP decreases cellular invasion and migration and increases DNA repair in MDA-MB-231. a (Left), Relative migration at 9 and 12 h of MDA-MB-231 cells transduced with doxycycline-inducible (96 h) lentiviral vector expressing a shRNA that targets both CtBP1 and CtBP214. (Right), Wound closure assay of MDA-MB-231 cells pre-treated 24 h with 10 μM CtBP inhibitors. The inset shows relative migration at 6 h of incubation. (*indicates P < 0.05). b Fluorescent images (left) and graph (right) of invading and migrating MDA-MB-231 cells on DAPI stained membranes following 24-h treatment of cells with vehicle or 10 μM CtBP inhibitors during invasion and migration in Corning® Biocoat™ Matrigel® Invasion Chambers. All experiments represent the average and standard error of at least two biological replicates with each experiment performed at least twice. The error bars represent the s.d. of the mean from at least two independent experiments. P-values are calculated from the Student’s T-Test relative to the vehicle control. *Indicates P < 0.05 and **indicates P < 0.01. c (Top) Phospho-gamma H2AX foci profile of cells following ionizing radiation (5 Gy) and recovery at 0, 12 and 24 h. d Relative rate of DNA repair is expressed as total percent of nuclei containing two or more phospho-gamma H2AX foci per high power field. The error bars represent the s.d. of the mean from at least two independent experiments. P-values are calculated from the Students T-Test relative to the vehicle control. *Indicates P < 0.05 and **indicates P < 0.01

Nuclear CtBP and LSD1 associated pathways in breast breast cancer patients were quantitively stratified cancer patients according to CtBP1, CtBP2, and LSD1 nuclear expression Previous studies have shown that patient breast cancer into low, medium and high terciles of expression (see samples that co-express high levels of LSD1 and histone Materials and methods). Notably, within this cohort, the deacetylases (SIRT1, HDAC2) have decreased survival45. level of CtBP1 or CtBP2 did not differ greatly in patients To evaluate the possible significance of high nuclear levels between breast cancer subtypes or estrogen receptor of CtBP and LSD1 in breast cancer survival, we utilized a status. In fact, slightly higher levels were demonstrated in unique resource of patient samples in which nuclear, estrogen-receptor-positive subtypes as opposed to estro- CtBP1, CtBP2, and LSD1 have been quantitatively deter- gen receptor-negative subtypes (Supplementary Fig. 3). mined and compared to paired gene expression data These categories were then used to group patients, based on RNA-seq analysis (Material and methods, also according to their CtBP1, CtBP2 and LSD1 nuclear see Supplementary Fig. 3). Using this available data, 126 expression, in to three categories: (I) CtBP1/LSD1 = Low:

Official journal of the Cell Death Differentiation Association Byun et al. Cell Death and Disease (2019) 10:689 Page 9 of 15

A B Veh Par 100 CI24 CI24+Par

6 OVOL2 GATA3 * 80 FOXA1 GRHL2 CI24 60 Veh 4 * * Par ** ** * * * * * * ** * 40 * * * * ** * * * 2 * * * * *

(normalized to 18s) 20 CI24 + Par -PCR mRNA Fold Change -PCR mRNA

qRT 0 0

2 3 r r Density v1.0 (Percent) Relative Wound 0 5 10 15 20 25 h 19 2 2 24 M e I Pa V C CI CI CI +Par m 9+ 3 4+Pa .5 I1 I22+ParI2 I2 2 Time (hours) C C C C r Pa

CDFACTOR 100 Tris Normalized Es te Std. Error t-value p-value CI24 Time 3.866 0.25 15.437 3.17E-15 80 Par CI24+Par CI24 -5.581 5.059 -1.103 0.279 Tris 60 Par -13.131 5.059 -2.595 0.0148

40 CI24 : Par -12.418 5.059 -2.454 0.0205 CI24 : Time -0.416 0.354 -1.174 0.249 20 Par : Time -1.461 0.354 -4.125 0.0002

0 CI24 : Par : Time -2.604 0.354 -7.351 5.27E-08 Relative Wound Density v1.0 (Percent) Wound Relative 0 1 10 Time (Hours)

Fig. 7 Pharmacologic inhibition of CtBP potentiates LSD1 inhibition to depress pro-epithelial gene expression and decrease breast cancer cell migration. a Relative expression of the pro-epithelial genes OVOL2, GATA3, FOXA1, and GRHL2 in MDA-MB-231 cells treated with vehicle alone, 2.5 mM pargyline alone, CI24 inhibitor alone or in combination with 2.5 mM pargyline and 10 μM CtBP inhibitors. The error bars represent the s.d. of the mean derived from at least two independent experiments. P-values are calculated from the Student’s T-Test relative to the vehicle control. *Indicates P < 0.05 and **indicates P < 0.01. b IncuCyte ZOOM™ assay of MDA-MB-231 cell migration in cells treated with either vehicle, 10 μM CI24 CtBP inhibitor, 2.5 mM pargyline, or 2.5 mM pargyline with 10 μM CI24. The error bars represent the s.d. of the mean derived from at least two independent experiments *indicates P < 0.05 and **indicates P < 0.01. c Logistic regression modeling of the dependence of cell mobility (wound density) on time and added drug(s) based on data plotted in (b). Hours are shown in log scale. d Tabulation of values of the multivariate analysis based on logistic analysis of the single and combined influence of time, CI24, pargyline, and the combination of pargyline and CI24 on wound density shown in (c). P-values indicate the significance of the strength of interaction of the combined conditions with respect to their influence on cell mobility

Low = low; (II) CtBP1:LSD1, medium:medium = medium; gene pathways enriched in the genes differentially and (III) CtBP1/LSD1 = high:high = high (Fig. 8a). The gene expressed by ANOVA of the three stratifications show expression of the patients in these three categories were significant differences (Fig. 8c–e). CtBP1/LSD1 differen- then assessed by analysis of variance (ANOVA) (Fig. 8a) to tially expressed genes show significant enrichment for identify genes that were differentially expressed between signaling, CtBP1/CtBP2 differential the three groups. Examples of differentially expressed expressed genes are enriched for immune response genes genes are shown by boxplot in Fig. 8a. Notably, many including immune checkpoint regulators (Fig. 8d), and genes linked to chromatin modification and LSD1 only differentially expressed genes are enriched are enriched in the genes differentially expressed in these with genes associated with regulation of the extracellular categories (Fig. 8a, and Supplementary Tables 1–4). Simi- matrix (Fig. 8e). Unique genes identified by ANOVA larly, genes that are differentially expressed based on the profiling of differentially expressed genes across 5 cate- stratification of CtBP1/CtBP2 or LSD1, respectively, were gories of CtBP1:LSD1 combinations (including CtBP1: identified (Supplementary Table 1). A Venn diagram of LSD1 = Low/High and CtBP1:LSD1 = High/Low) was not the genes common to all three stratifications is shown in additionally informative (Fig. S4 and Supplementary Fig. 8b and demonstrates very little overlap. Moreover, the Tables 1 and 5).

Official journal of the Cell Death Differentiation Association Byun et al. Cell Death and Disease (2019) 10:689 Page 10 of 15

A Nuclear CtBP1/LSD1: low medium high

0.0072 0.00086 0.0047 0.033 0.35 0.012 6 800 10.0 0.22 0.72 0.095 0.0034 0.18 0.0079 0.095 0.035 200 0.34 0.98 4 0.46 0.15

l l l 600 7.5 10 l 4 150 l 3 400 5.0 ERBB2 CDK5 MBD4

2 WHSC1 100 l TIMP3 Anova, p = 0.027 EP300 5 2 Anova, p = 0.047 l l 200 50 1 2.5

l

Anova, p = 0.038 l 0 Anova, p = 0.003 0 0 0 0.0 Anova, p = 0.025 0 Anova, p = 0.018

0.0012 0.0087 0.046 0.0047 20 0.0041 12 0.014 6 0.37 0.033 0.18 0.1 0.53 0.81 0.6 0.0028 0.75 0.55 30 0.031 0.31 0.046 0.0048 l 15 9 l

l

4 l 0.4 0.50 20 6 l 10

Anova, p = 0.0033 INO80B KDM4D JMJD4 GPR1

IGF1R Anova, p = 0.027

ADGRV1 Anova, p = 0.0026

2 l l 0.25 10 0.2 3 l 5 l

0 0.00 0 Anova, p = 0.0017 0.0 0 Anova, p = 0.0039 0 Anova, p = 0.011 l

BCLSD1/CtBP1 Entities Pathway name found ratio p-value CtBP/LSD1 CtBP1/CtBP2 GRB7 events in ERBB2 signaling 3 / 6 4.27e-04 0.009 ERBB2 Activates PTK6 Signaling 5 / 18 0.001 0.009 66 690 892 ERBB2 Regulates Cell Motility 5 / 19 0.001 0.011 GRB2 events in ERBB2 signaling 5 / 20 0.001 0.014 12 Intestinal infectious diseases 2 / 3 2.13e-04 0.019 161 41 Downregulation of ERBB4 signaling 3 / 10 7.11e-04 0.033 BH3-only proteins associate with and inactivate anti-apoptotic BCL-2 3 / 11 7.82e-04 0.042 758 members SHC1 events in ERBB4 signaling 4 / 20 0.001 0.052

LSD1 Mitochondrial transcription 1 / 1 7.11e-05 0.067 termination PI3K events in ERBB2 signaling 4 / 22 0.002 0.069

CtBP1/CtBP2 Entities DELSD1 Entities Pathway name found ratio p-value Pathway name found ratio p-value Interleukin-10 signaling 16 / 86 0.006 0.001 Interleukin-18 signaling 5 / 11 7.82e-04 0.001 Interleukin-2 signaling 5 / 14 9.96e-04 0.005 Insulin-like Growth Factor-2 mRNA Binding Proteins 5 / 13 9.25e-04 0.003 DAP12 signaling 8 / 35 0.002 0.007 (IGF2BPs/IMPs/VICKZs) bind RNA GPVI-mediated activation cascade 9 / 43 0.003 0.007 cell surface interactions 14 / 86 0.006 0.006 IRAK4 deficiency (TLR5) 3 / 5 3.56e-04 0.007 Collagen degradation 12 / 69 0.005 0.006 Diseases of Immune System 7 / 34 0.002 0.018 Assembly of collagen fibrils and other multimeric structures 11 / 67 0.005 0.012 Diseases associated with the TLR 7 / 34 0.002 0.018 signaling cascade Extracellular matrix organization 36 / 329 0.023 0.014 MyD88 deficiency (TLR5) 2 / 3 2.13e-04 0.023 Collagen chain trimerization 8 / 44 0.003 0.018 PD-1 signaling 8 / 45 0.003 0.026 RUNX2 regulates genes involved in cell migration 4 / 14 9.96e-04 0.021 Nef and 3 / 9 6.40e-04 0.034 NFG and proNGF binds to p75NTR 2 / 3 2.13e-04 0.021 Syndecan interactions 6 / 29 0.002 0.022

Fig. 8 Inferred gene targets and pathway vulnerabilities associated with combined nuclear enrichment of CtBP1 and LSD1 in breast cancer patients. a Boxplot displays representative of genes that showed significant differential expression by ANOVA analysis (p-value < 0.05) across the three breast cancer patient categories stratified by low, medium, and high nuclear co-expression of CtBP1 and LSD1 using a quantitative immune-histochemical analysis of nuclear staining for CtBP1, and LSD1 based on analysis of the RNA-seq profiles from each patient in each class (see materials and methods). ANOVA test was applied to identify which genes differed significantly across the three categories. (p-values for paired categories are also shown). b Venn diagram showing the overlap between genes there were significantly differentially expressed in the categories: CtBP1/LSD1, CtBP1/CtBP2, and LSD1. c Pathway analysis showing significantly enriched pathway genes that are differentially expressed in association with CtBP1/LSD1 nuclear expression. d Pathway analysis showing significantly enriched pathway genes that are differentially expressed in association with CtBP1/CtBP2 nuclear expression. e Pathway analysis showing significantly enriched pathway genes that are differentially expressed in association with LSD1 nuclear expression alone

Official journal of the Cell Death Differentiation Association Byun et al. Cell Death and Disease (2019) 10:689 Page 11 of 15

Discussion with the development of 2nd or 3rd generation CtBP In summary, using computer-assisted drug design to inhibitors that act with higher affinity. Currently, the most screen for possible inhibitors of CtBP function, we have potent compound appears to be CI24 which shows the identified four lead compounds that, through disruption highest uniform correlation in potency of transcriptional of CtBP dimerization, reactivate CtBP-repressed genes. de-repression, with eviction of chromatin modifiers, The mechanisms underlying this inhibitory activity are influences on cell migration/invasion, and increase in associated with the ability of this class of compounds to DNA repair. The provocative finding that differential disrupt the CtBP-mediated recruitment of multiple dif- enrichment of CtBP1/LSD1 shows selective influence on ferent chromatin-modifying complexes to targeted genes. ERRB2/4 signaling (Fig. 8c) suggests a broader application These include the , HDAC1; and the of this targeting strategy beyond TNBC subtypes. More- histone demethylase, LSD1. These findings not only over, it suggests additional opportunities for combined implicate an important application of these compounds in therapy. An important aspect of this current study is the strategies for therapeutic intervention but also demon- correlation of nuclear levels of CtBP1 and LSD1 with gene strate their utility as investigative tools to define epige- expression patterns in patient samples. Although cell lines netic gene regulatory pathways and mechanisms linked to are an easily tractable system that led to the cell-type CtBP. The application of these compounds as investiga- specific identification of GATA3, FOXA1, and OVOL2 as tive tools also provides a proof of principle for the com- major regulatory targets of CtBP, these genes do not show binatorial use of CtBP inhibitors with other epigenetically up as top targets in patient samples (Fig. 8). This reflects targeted compounds or therapeutic strategies. The com- the innate heterogeneity and plasticity of patient breast bined use of LSD1 and CtBP inhibitors offers a new cancer tissues and also is consistent with the significant concept in this therapeutic approach. cell type-specificity of the influence of CtBP inhibition, The current rapid expansion of novel strategies to target mentioned previously (Fig. 4), and observed in CtBP- both epigenetic regulatory enzymes and epigenetic reg- depleted cell lines14. Other notable correlations found in – ulatory complexes46 48 suggests a prominent emerging patient samples is the high significance of the DNA repair new role for these compounds and other recently char- genes ATM, LIG4, PARP3, SETX, and GRF2H4 genes acterized CtBP inhibitors21,22 in combinatorial strategies revealed by ANOVA analysis of the CtBP1/CTBP2 high for epigenetic intervention. Although these compounds category (see Supplementary Table 1). In summary, the bind with modest affinity (in the micromolar range), they findings in this study provide a new and novel conceptual serve as prototypes from which to bootstrap the design of groundwork for the development of more effective novel molecules with higher binding affinity and activity treatment strategies and introduce a novel class of com- toward CtBP-regulated pathways. Similarly, although pounds to be explored in combinatorial strategies to pargyline shows measurable synergy with CtBP inhibitors, exploit vulnerabilities in the epigenetic regulation of its primary use in clinical trials has been associated with breast cancer. treatments for hypertension. Nonetheless, there is renewed interest in therapeutic targeting of LSD1 as Materials and methods several new compounds that disrupt LSD1 function Cell lines and constructs including: GSK2879552, ORY-100, 4SC-202, IMG-7289, MDA-MB-231 and MCF-7 cells were purchased from INCB-59872, and derivatives, are being ATCC. All starting cultures were screened for myco- assessed in clinical trials for activity against leukemia43. plasma contamination approximately 4–5 months prior to This is particularly relevant given the recent finding that use, and experiments were conducted on cell lines grown loss of LSD1 is associated with an enhanced anti-tumor for no more than 13–15 passages. Short hairpin sequences response through upregulation of double-stranded RNA for RNAi depletion of CtBP1 and CtBP2 were cloned into stress response secondary to lost repression of endogen- the doxycycline-inducible (Tet-ON) pINDUCER lentiviral ous retroviral elements49. Furthermore, analysis of TCGA vector system that produces a miR30-based short hairpin data show an inverse relationship between CD8+ T-cell that targets both CtBP1 and CtBP213,50. CFP-CTBP1 infiltration and the RNA levels of LSD149. It is not clear and YFP-CTBP2 plasmids were provided by Dr. Jermey P. what underlies the cell-type specific dose sensitivity we Blaydes51. have observed in the current study, however, this has been observed with other CtBP inhibitors10. Whether or not it Cell culture and tissues reflects sensitivity thresholds based on the level of epi- MDA-MB-231 and MCF-7 cells were maintained in thelial differentiation in breast cancer will require exten- regular DMEM supplemented with 10% (v/v) FBS, sive investigation beyond the scope of this initial proof-of- penicillin-streptomycin (Invitrogen) and insulin as pre- principle report. Such issues may become better resolved viously described14.

Official journal of the Cell Death Differentiation Association Byun et al. Cell Death and Disease (2019) 10:689 Page 12 of 15

Computer-assisted drug design photobleaching, respectively. Each sample was scored for ChemNavigator iResearch Library from Sigma Aldrich24 greater than 15 nuclei in order to achieve statistical sig- was searched for molecules containing oxaldehydic acid nificance. 15 regions of interest were randomly measured structures similar to MTOB or structurally similar pro- within each nucleus. A FRET signal was considered be panedioic, 3-oxobutanoic, and 4-oxopropanoic acid. positive if the FRET efficiency values (EF) obtained in the These moieties can form favorable interactions with three experiment exceeded those of random FRET in the residues (His315, Glu295, Arg 266) demonstrated to negative control. function as a catalytic triad in the active site of CtBP in addition to other essential residues at the catalytic site, Chromatin immunoprecipitation and immunoprecipitation including Arg266 and Arg678. Quantitative structure- ChIP was performed as previously described53 and the activity relationship (QSAR) models were generated using list of primers and antibodies used in the study are pro- MTOB and a series of 11 alpha-keto acids with measured vided in the Supplementary materials and methods. kinetic properties for the catalytic domain of CtBP. The best model obtained was then applied against the set of Western blotting RNA isolation, and RT-qPCR selected acidic compounds from ChemNavigator, and RNA isolation from cell lysates and RT-qPCR were compounds with high predicted substrate activity values performed as described previously53 and normalized to were filtered for drug-like properties using the Lipinski 18S rRNA. The total RNA was prepared using the Rule52. Filtered candidates selected by the QSAR model- RNAeasy kit (Qiagen) following the manufacturer’s pro- ing were then further evaluated by docking into the tocol. The RNA was quantified and 1 μg of total RNA was published crystallographic structure of the CtBP dehy- used for each reverse transcription. Reverse transcription drogenase core with NAD+ and acetic acid8. Docking was was carried out by following the QuantiTect® Reverse done using Glide (27) and the docked compound struc- Transcription procedure (Qiagen). For western blotting, tures were required to be stabilized by at least two the cells were collected and resuspended in RIPA lysis hydrogen bonds to Arg266, Gly101 or His315 in the CtBP buffer (50 mM Tris pH 7.5, 1 mM EDTA, 150 mM NaCl, active site. This screen identified 31 compounds of which 0.1% SDS, 1% TritonX-100, 1% sodium deoxycholate and 24 were commercially available. (See virtual screening freshly added proteinase inhibitor cocktail) for 30 min on methods in Supplementary materials and methods) ice. The lysates were centrifuged for 20 min at 12,000 rpm and the supernatants were used for quantitation and Acceptor photobleaching FRET western blotting. Nuclear extracts were prepared and Expression vectors for CFP-CtBP1 (pSCFP3A) and YFP- analyzed as described previously53. CtBP2 (pSYFP2) were co-transfected into MDA-MB-231 wild-type cells using lipofectamine 2000 reagent (Thermo Invasion and migration assays Fischer Scientific). 2 × 104 cells/well were seeded into Invasion assay was performed using MDA-MB-231 cells Nunc Lab-Tek 8 chambered coverglass (Thermo Fischer with the Corning® Biocoat™ Matrigel® Invasion Chambers Scientific). Cells were then treated with either 100 μMof (cat. 354480). Replicates were carried out according to the Tris control vehicle or 10 μM of the four different CtBP manufacturer’s recommendation after 24 h of treatment Inhibitors (CI19, CI22, CI23, and CI24). We used a of Tris and 10 μM CtBP inhibitors (CI19, CI22, CI23, and plasmid encoding a CFP-YFP fusion protein with a 2-aa CI24). After 24 h of the invasion, the membranes were spacer between CFP and YFP as a positive control for washed in 1X PBS, fixated in methanol, stained with FRET29. Co-transfection with CFP-CtBP1 (pSCFP3A) and DAPI, and visualized. Wound closure assay was per- unconjugated YFP (pCMV6AC-mYFP) plasmids were formed using the Radius™ assay from Cell Biolabs, Inc. used as negative control. Cells were imaged with a 60 × 1.4 Cells were incubated 24 h with 10 μM CtBP inhibitors NA Zeiss immersion objective and ×2 zoom using 514 nm prior to initiation of the migration assay. For assays laser line of argon laser (25 mW) and 405 nm laser line of comparing cellular migration in response to combined Diode laser (30 mW) on LSM780 confocal microscope addition of drugs, cellular migration was measured using (Carl Zeiss, Inc, Thornwood, NY, USA) in the Optical the IncuCyte® ZOOM System, real-time, a quantitative Microscopy Core (NCI/CCR/LRBGE). YFP was photo- live-cell analysis system for high definition phase contrast bleached by scanning the whole cell 10 times using the images to monitor wound closer over time as directed by 514 laser line of an argon laser at 100% intensity. The the manufacturer’s instructions. efficiency of Fluorescence Resonance Energy Transfer (FRET) EF was measured by acceptor photobleaching Gamma H2A.X foci formation assay 29 method as described based on the equation: EF= MDA-MB-231 wild type and CtBP1/2 knockdown cells (ICFP, after − ICFP, before)/ICFP, after, where ICFP, after and ICFP, were seeded in 8 chamber slides at a density of before referring to CFP intensities after and before YFP 10,000 cells/well. MDA-MB-231 wild type cells were

Official journal of the Cell Death Differentiation Association Byun et al. Cell Death and Disease (2019) 10:689 Page 13 of 15

treated with the CtBP inhibitors (CI19, CI22, CI23, and each containing a third, i.e. 33.33% of a given population. CI24) and Tris Control for 24 h prior to Gamma Irra- The first tercile (low) represents the lowest 33.33% of the diation. MDA-MB-231 wild type and CtBP1/2 knock- data (1–33.33%); the second tercile represents the med- down cells were exposed to 5 Gy of Gamma irradiation. ium 33.33% value (33.34–66.66%) and third highest 33.33 After 0, 12, and 24 h post-Gamma irradiation, cells were value (66.34–100%). Based on those protein values, the washed three times with 1× PBS, fixed with 3.5% paraf- patients were classified into these three categories and ormaldehyde for 20 min followed by 70% cold ethanol defined them as low, medium and high. Finally, patients overnight. Cells were then washed three times with 1× were segregated into combined CtBP1: LSD1 categories of PBS, stained with anti-phospho-Histone H2A.X antibody Low: Low; Medium: Medium; and High: High and used (Ser139) (catalog # 05-636, Millipore) at 1/1000 primary for comparison of their gene expression patterns. antibody dilution for 1 h, washed three times with 1× PBS, followed by staining with secondary Alexa Fluor 488 RNA-seq goat anti-mouse antibody (Catalog # A11001, Life Following a review of H&E stained slides areas of tumor Technologies) at 1/2000 dilution for one hour. Cells were with >80% nuclei were circled, and 2.5 × 2–3 mm tissue then washed three times with 1× PBS followed by stain- cores were extracted from the corresponding regions of ing with 1/5000 DAPI dilution. Cells were then mounted FFPE tissue blocks for N = 126 patients. Sample cores were with anti-fade mounting media and covered with cover- shipped to BGI Beijing Genome Institute (BGI) for further slips. Different fluorescent images were taken with processing as previously described55,56. Briefly, the total Axiovert 200 M and only cells with 3 plus phospho-H2A RNA samples were first treated with DNase I, followed by foci were scored. an mRNA enrichment step enriched by using the oligo (dT) coupled magnetic beads. Following fragmentation, the Methods for immunohistochemistry first strand of cDNA was synthesized by using random Tissue microarray construction and scoring hexamer-primers. Buffer, dNTPs, RNase H and DNA Following IRB approval from East Carolina University polymerase I were added to synthesize the second strand. and the National Institutes of Health Intramural research The double-strand cDNA was purified with magnetic program, approximately 180 de-identified formalin-fixed beads and end reparation and 3’-end single nucleotide A and paraffin-embedded tissue from patients who were (adenine) addition was then performed. Finally, sequencing diagnosed and underwent surgery for Stage 0 to Stage IV adaptors were ligated to the fragments. The fragments breast cancer between 2001 and 2010 at Pitt County were enriched by PCR amplification. During the QC step, Memorial Hospital (now Vidant Medical Center), Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time Greenville, NC. Following a review of hematoxylin and PCR System were used to qualify and quantify the sample eosin-stained sections, regions of interest were outlined, library. The library was sequenced (60 M paired-end read and 1 mm cores were removed from corresponding blocks per sample) on an Illumina HiSeqTM4000. using a Pathology Devices TMArrayer (Westminster, MD). All arrays contained appropriately chosen positive Sequence data analysis and negative control tissue. Digital image analysis and After sequencing, the raw reads were filtered (BGI). scoring of IHC staining was performed using Leica Aperio Data filtering included removing adapter sequences, digital analysis platforms, in which three representative contamination and low-quality read from raw reads. The regions of tumor were outlined on each core by a cleaned reads (fastQ) were mapped to the reference pathologist and scored digitally using the Nuclear v9 sequence using HISAT57. Raw reads and RPKM for each algorithm (CtBP1, CtBP2, and LSD1) to generate a histo- sample were calculated using HOMER58. Differential gene score (H-Score; 0–300) based on the percent of positive expression was performed using EdgeR59. cells with assigned intensity thresholds of negative (0), low (1), moderate (2) or high (3) as previously described54. Statistical analysis Breast tumor tissue microarrays were stained, using A Spearman rank correlation test was performed to test monoclonal primary antibodies, CTBP1 and CTBP2 the relation between its protein H-score and gene (33871, 39008 Millipore) at 1:100,000 overnight with high expression (RPKM value) values60. A completely unsu- pH (Catalog No. M361201-2, DAKO). LSD1 (Abcam, pervised hierarchical clustering approach was performed ab129195) staining was done at 1:2000 overnight with low on all 486 breast samples of the protein data set. pH (Catalog No. MA5-13191, DAKO). Using the assigned Complete linkage and distance correlations were used scale of 1–300, patients were then classified into terciles for clustering protein data with bootstrap resampling based on the H-Scores for CtBP1, CtBP2, and LSD1, techniques. The stability of the clustering was estimated respectively. The data was then categorized into terciles with the ‘pvclust’ Rpackage61 available on CRAN based on the divided ordered distribution into three parts, http://cran.rproject.org/web/packages/ pvclust/). A two-

Official journal of the Cell Death Differentiation Association Byun et al. Cell Death and Disease (2019) 10:689 Page 14 of 15

sided t-test was employed to test the null hypothesis (H0) 4. Dcona, M. M., Morris, B. L., Ellis, K. C. & Grossman, S. R. CtBP- an emerging assumption of equality of the protein values in two and novel small molecule drug target: advances in the under- fi standing of its oncogenic action and identification of therapeutic inhibitors. de ned groups of data and demonstrated by violin plots Cancer Biol. Ther. 18,379–391 (2017). 62 using R software and ggplot2 package . ANOVA and 5. Deng, Y. et al. Redox-dependent Brca1 transcriptional regulation by an NADH- logistic regression analysis were conducted using R sta- sensor CtBP1. Oncogene 29,6603–6608 (2010). 6. Shi, Y. et al. Coordinated histone modifications mediated by a CtBP co- tistical analysis software. A p-value thresholds 0.05 was repressor complex. Nature 422,735–738 (2003). applied to select the top significant genes (Supplementary 7. Chinnadurai, G. CtBP, an unconventional transcriptional in Table 1). A reactome open-source browser (version 3.6), development and oncogenesis. Mol. Cell 9,213–224 (2002). 8. Kumar, V. et al. Transcription corepressor CtBP is an NAD(+)-regulated with curated and peer-reviewed pathway database (reac- dehydrogenase. MolCell 10,857–869 (2002). tome release 67) was used for pathway analysis of the top 9. Zhang, Q., Piston, D. W. & Goodman, R. H. Regulation of corepressor function genes identified (https://reactome.org/PathwayBrowser/). by nuclear NADH. Science 295,1895–1897 (2002). 10. Straza, M. W. et al. Therapeutic targeting of C-terminal binding protein in human cancer. Cell Cycle 9,3740–3750 (2010). Acknowledgements 11. Bhambhani, C., Chang, J. L., Akey, D. L. & Cadigan, K. M. The oligomeric state of This work was supported by the intramural research programs of the National CtBP determines its role as a transcriptional co-activator and co-repressor of Cancer Institute and the National Institute on Minority Health and Health Wingless targets. EMBO J. 30,2031–2043 (2011). Disparities, Bethesda Maryland, 20892 and support from the Cancer Center 12. Thio,S.S.,Bonventre,J.V.&Hsu,S.I.The CtBP2 co-repressor is regulated by Support Grant (P30CA013696). This work was supported in part with Federal NADH-dependent dimerization and possesses a novel N-terminal repression funds from the Frederick National Laboratory for Cancer Research, National domain. Nucleic Acids Res 32, 1836–1847 (2004). Institutes of Health, under contract HHSN261200800001E. The content of this 13. Di,L.J.,Fernandez,A.G.,DeSiervi,A.,Longo,D.L.&Gardner,K.Transcriptional publication does not necessarily reflect the views or policies of the regulation of BRCA1 expression by a metabolic switch. Nat.Struct.Mol.Biol.17, Department of Health and Human Services, nor does mention of trade names, 1406–1413 (2010). commercial products or organizations imply endorsement by the US 14. Di, L. J. et al. Genome-wide profiles of CtBP link metabolism with genome Government. stability and epithelial reprogramming in breast cancer. Nat. Commun. 4,1449 (2013). Author details 1 15. Byun, J. S. & Gardner, K. C-Terminal binding protein: a molecular link between National Institute on Minority Health and Health Disparities, Bethesda, MD metabolic imbalance and epigenetic regulation in breast cancer. Int J. Cell Biol. 20892, USA. 2Genetics Branch, National Cancer Institute, Bethesda, MD 20892, 3 2013, 647975 (2013). USA. Laboratory of Cancer Biology and Genetics, National Cancer Institute, 16. Grooteclaes, M. et al. C-terminal-binding protein corepresses epithelial and Bethesda, MD 20892, USA. 4Laboratory of Pathology, National Cancer Institute, 5 proapoptotic gene expression programs. Proc. Natl Acad. Sci. USA 100, Bethesda, MD 20892, USA. Chemical Biology Laboratory, Center for Cancer – 6 4568 4573 (2003). Research, National Cancer Institute, Frederick, MD 20892, USA. Basic Science 17. Juo, Y. Y. et al. Epigenetic therapy for solid tumors: from bench science to Program, Chemical Biology Laboratory, Leidos Biomedical Research Inc., clinical trials. Epigenomics 7, 215–235 (2015). Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA. 7 8 18. Achouri, Y., Noel, G. & Van, S. E. 2-Keto-4-methylthiobutyrate, an intermediate National Institute, Bethesda, MD 20892, USA. Brody School in the methionine salvage pathway, is a good substrate for CtBP1. Biochem- of Medicine at East Carolina University, Greenville, NC 27834, USA. – 9 Biophys Res. Commun. 352,903 906 (2007). Department of Pathology and Cell Biology, Columbia University Medical 19. Birts, C. N. et al. A cyclic peptide inhibitor of C-terminal binding protein 10 ı Center, New York, NY 10032, USA. Laboratorio de Oncolog a Molecular y dimerization links metabolism with mitotic fidelity in breast cancer cells. Chem. ı Nuevos Blancos Terapeuticos, Instituto de Biolog a y Medicina Experimental Sci. 4,3046–3057 (2013). (IBYME), CONICET, Buenos Aires, Argentina 20. Hilbert,B.J.,Grossman,S.R.,Schiffer,C.A.&Royer,W.E.Jr.Crystalstructuresof human CtBP in complex with substrate MTOB reveal active site features useful Conflict of interest for inhibitor design. FEBS Lett. 588,1743–1748 (2014). The authors declare that they have no conflict of interest. 21. Hilbert, B. J. et al. Structure-guided design of a high affinity inhibitor to human CtBP. ACS Chem. Biol. 10, 1118–1127 (2015). 22. Korwar, S. et al. Design, synthesis, and biological evaluation of substrate- Publisher’s note competitive inhibitors of C-terminal binding protein (CtBP). Bioorg. Med Chem. Springer Nature remains neutral with regard to jurisdictional claims in 24,2707–2715 (2016). published maps and institutional affiliations. 23. May, T. et al. BRCA1 expression is epigenetically repressed in sporadic ovarian cancer cells by overexpression of C-terminal binding protein 2. Neoplasia 15, – Supplementary Information accompanies this paper at (https://doi.org/ 600 608 (2013). 10.1038/s41419-019-1892-7). 24. Weidlich, I. E. et al. Inhibitors of human tyrosyl-DNA phospodiesterase (hTdp1) developed by virtual screening using ligand-based pharmacophores. Bioorg. Med Chem. 18,182–189 (2010). Received: 28 March 2019 Revised: 17 July 2019 Accepted: 8 August 2019 25. Meyer, K. B. & Carroll, J. S. FOXA1 and breast cancer risk. Nat. Genet 44, 1176–1177 (2012). 26. Roca, H. et al. Transcription factors OVOL1 and OVOL2 induce the mesenchymal to epithelial transition in human cancer. PLoS ONE 8, e76773 (2013). References 27. Bernardo, G. M. et al. FOXA1 is an essential determinant of ERalpha expression 1. Boyd, J. M. et al. A region in the C-terminus of adenovirus 2/5 E1a protein is and mammary ductal morphogenesis. Development 137,2045–2054 (2010). required for association with a cellular phosphoprotein and important for the 28. Cieply, B. et al. Suppression of the Epithelial-Mesenchymal Transition by negative modulation of T24-ras mediated transformation, tumorigenesis and Grainyhead-like-2. Cancer Res. 72,2440–2453 (2012). – metastasis. EMBO J. 12,469 478 (1993). 29. Karpova, T. S. et al. Fluorescence resonance energy transfer from cyan to 2. Chinnadurai, G. The transcriptional corepressor CtBP: a foe of multiple tumor yellow fluorescent protein detected by acceptor photobleaching using con- – suppressors. Cancer Res 69, 731 734 (2009). focal microscopy and a single laser. J. Microsc 209(Pt 1), 56–70 (2003). 3. Byun,J.S.&Gardner,K.C-Terminalbinding protein: a molecular link between 30. Wu, S., Luo, Z., Yu, P. J., Xie, H. & He, Y. W. Suberoylanilide hydroxamic acid metabolic imbalance and epigenetic regulation in breast cancer. Int. J. Cell Biol. (SAHA) promotes the epithelial mesenchymal transition of triple negative 2013, 14 (2013). breast cancer cells via HDAC8/FOXA1 signals. Biol. Chem. 397,75–83 (2016).

Official journal of the Cell Death Differentiation Association Byun et al. Cell Death and Disease (2019) 10:689 Page 15 of 15

31. Kong,S.L.,Li,G.,Loh,S.L.,Sung,W.K.&Liu,E.T.Cellularreprogrammingby 46. Wang, J. Q. & Wu, K. J. Epigenetic regulation of epithelial-mesenchymal the conjoint action of ERalpha, FOXA1, and GATA3 to a ligand-inducible transition by hypoxia in cancer: targets and therapy. Curr.Pharm.Des.21, growth state. Mol. Syst. Biol. 7, 526 (2011). 1272–1278 (2015). 32. Grooteclaes,M.L.&Frisch,S.M.Evidence for a function of CtBP in epithelial 47. Byler,S.etal.Geneticandepigeneticaspects of breast cancer progression and gene regulation and anoikis. Oncogene 19, 3823–3828 (2000). therapy. Anticancer Res. 34,1071–1077 (2014). 33. Deng, Y. et al. Transcriptional down-regulation of Brca1 and E-cadherin by 48. Ahuja, N., Easwaran, H. & Baylin, S. B. Harnessing the potential of epigenetic CtBP1 in breast cancer. Mol. Carcinog. 51, 500–507 (2012). therapy to target solid tumors. J. Clin. Investig. 124,56–63 (2014). 34. Kuppuswamy, M. et al. Role of the PLDLS-binding cleft region of CtBP1 in 49. Sheng, W. et al. LSD1 ablation stimulates anti-tumor immunity and enables recruitment of core and auxiliary components of the corepressor complex. checkpoint blockade. Cell 174,549–563.e519 (2018). Mol. Cell Biol. 28,269–281 (2008). 50. Meerbrey, K. L. et al. The pINDUCER lentiviral toolkit for inducible RNA inter- 35. Subramanian, T. & Chinnadurai, G. Association of class I histone deacetylases ference in vitro and in vivo. Proc. Natl. Acad. Sci. USA 108, 3665–3670 (2011). with transcriptional corepressor CtBP. FEBS Lett. 540,255–258 (2003). 51. Birts CN, et al. A cyclic peptide inhibitor of C-terminal binding protein 36. Wang, Y. et al. LSD1 Is a subunit of the NuRD complex and targets the dimerization. Chem. Sci. 10, 3046–3057. metastasis programs in breast cancer. Cell 138, 660–672 (2009). 52. Lipinski, C. A., Lombardo, F., Dominy, B. W. & Feeney, P. J. Experimental and 37. Redon, C. E. et al. gamma-H2AX and other histone post-translational mod- computational approaches to estimate solubility and permeability in drug ifications in the clinic. Biochim. Biophys. Acta. 1819, 743–756 (2012). discovery and development settings. Adv. Drug Deliv. Rev. 46,3–26 (2001). 38. Boulding, T. et al. LSD1 activationpromotesinducibleEMTprogramsand 53. Byun, J. S. et al. ELL facilitates RNA polymerase II pause site entry and release. modulates the tumour microenvironment in breast cancer. Sci. Rep. 8,73 Nat. Commun. 3,633(2012). (2018). 54. McCarty, K. S. Jr., Miller, L. S., Cox, E. B., Konrath, J. & McCarty, K. S. Sr Estrogen 39. Vasilatos, S. N. et al. Crosstalk between lysine-specific demethylase 1 (LSD1) receptor analyses. Correlation of biochemical and immunohistochemical and histone deacetylases mediates antineoplastic efficacy of HDAC inhibitors methods using monoclonal antireceptor antibodies. Arch. Pathol. Lab Med in human breast cancer cells. Carcinogenesis 34,1196–1207 (2013). 109,716–721 (1985). 40. Pollock, J. A., Larrea, M. D., Jasper, J. S., McDonnell, D. P. & McCafferty, D. G. 55. Jia, W. et al. SOAPfuse: an algorithm for identifying fusion transcripts from Lysine-specific histone demethylase 1 inhibitors control breast cancer pro- paired-end RNA-Seq data. Genome Biol. 14, R12 (2013). liferation in ERalpha-dependent and -independent manners. ACS Chem. Biol. 7, 56. Peng, Z. et al. Comprehensive analysis of RNA-Seq data reveals extensive RNA 1221–1231 (2012). editing in a human transcriptome. Nat. Biotechnol. 30, 253 (2012). 41. Lynch, J. T., Harris, W. J. & Somervaille, T. C. LSD1 inhibition: a therapeutic 57. Kim,D.,Langmead,B.&Salzberg,S.L.HISAT:afastsplicedalignerwithlow strategy in cancer? Expert Opin. Ther. Targets 16, 1239–1249 (2012). memory requirements. Nat. Methods 12,357–360 (2015). 42. Huang,Y.,Vasilatos,S.N.,Boric,L.,Shaw,P.G.&Davidson,N.E.Inhibitorsof 58. Heinz, S. et al. Simple combinations of lineage-determining transcription histone demethylation and histone deacetylation cooperate in regulating factors prime cis-regulatory elements required for macrophage and B cell gene expression and inhibiting growthinhumanbreastcancercells.Breast identities. Mol. Cell 38,576–589 (2010). Cancer Res Treat. 131, 777–789 (2012). 59. Robinson, M. D., McCarthy, D. J. & Smyth,G.K.edgeR:aBioconductorpackage 43. Hosseini, A. & Minucci, S. A comprehensive review of lysine-specificdeme- for differential expression analysis of digital gene expression data. Bioinfor- thylase 1 and its roles in cancer. Epigenomics 9,1123–1142 (2017). matics 26,139–140 (2010). 44. Culhane, J. C., Wang, D., Yen, P. M. & Cole, P. A. Comparative analysis of small 60. Myers,J.L.,Well,A.&Lorch,R.F.Research design and statistical analysis.3rdedn molecules and histone substrate analogues as LSD1 lysine demethylase (Routledge, New York, 2010). inhibitors. J. Am. Chem. Soc. 132,3164–3176 (2010). 61. Suzuki, R. & Shimodaira, H. Pvclust: an R package for assessing the uncertainty 45. Derr, R. S. et al. High nuclear expression levels of histone-modifying enzymes in hierarchical clustering. Bioinformatics 22, 1540–1542 (2006). LSD1, HDAC2 and SIRT1 in tumor cells correlate with decreased survival and 62. Hintze, J. L. & Nelson, R. D. Violin plots: a box plot-density trace synergism. Am. increased relapse in breast cancer patients. BMC cancer 14, 604 (2014). Stat. 52, 181–184 (1998).

Official journal of the Cell Death Differentiation Association