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Table S2. Results of epigenetic library screen.

Drug screens leading to successful targeted therapies in cancer have been mainly based on cell viability assays identifying inhibitors of dominantly acting oncogenes. In contrast, there has been little success in discovering targeted therapies that reverse the effects of inactivating mutations in tumor-suppressor genes. BAP1 is one such tumor suppressor that is frequently inactivated in a variety of cancers, including uveal melanoma, renal cell carcinoma, and mesothelioma. Because BAP1 is an epigenetic transcriptional regulator of developmental genes, we designed a two-phase drug screen involving a cell-based rescue screen of transcriptional repression caused by BAP1 loss, followed by an in vivo screen of lead compounds for rescue of a BAP1-deficient phenotype with minimal toxicity in Xenopus embryos. The first screen identified 9 compounds, 8 of which were HDAC inhibitors. The second screen eliminated all except one compound due to inefficacy or toxicity. The resulting lead compound, quisinostat, has a distinctive activity spectrum, including high potency against HDAC4, which was recently shown to be a key target of BAP1. Quisinostat was further validated in a mouse model and found to prevent the growth of BAP1-mutant uveal melanomas. This innovative strategy demonstrates the potential for identifying therapeutic compounds that target tumor-suppressor mutations in cancer.Implications:Few drugs have been identified that target mutations in tumor suppressors. Using a novel 2-step screening approach, strategy, we identified quisinostat as a candidate for therapy in BAP1-mutant uveal melanoma. HDAC4 is implicated as a key target in uveal melanoma and perhaps other BAP1-mutant cancers.

S1. Doxycycline-inducible shRNA knockdown of BAP1 results in robust decrease of GLO1 expression in uveal melanoma cell lines. S2. Phenotypic rescue and toxicity analysis of three HDAC inhibitors in Xenopus embryos injected with control morpholino CtrlMO or bap1-targeting morpholino BAP1MO. S3. Quisinostat and trametinib combination exhibits mostly additive cytotoxic effect in uveal melanoma in vitro.

Table S1. Results of differential expression analysis

Bromodomain and extraterminal inhibitors (BETi) are promising cancer therapies, yet prominent side effects of BETi at effective doses have been reported in phase I clinical trials. Here, we screened a panel of small molecules targeting epigenetic modulators against human metastatic melanoma cells. Cells were pretreated with or without ascorbate (vitamin C), which promotes DNA demethylation and subsequently changes the sensitivity to drugs. Top hits were structurally unrelated BETi, including JQ1, I-BET151, CPI-203, and BI-2536. Ascorbate enhanced the efficacy of BETi by decreasing acetylation of histone H4, but not H3, while exerting no effect on the expression of BRD proteins. Histone acetyltransferase 1 (HAT1), which catalyzes H4K5ac and H4K12ac, was downregulated by ascorbate mainly via the TET-mediated DNA hydroxymethylation pathway. Loss of H4ac, especially H4K5ac and H4K12ac, disrupted the interaction between BRD4 and H4 by which ascorbate and BETi blocked the binding of BRD4 to acetylated histones. Cotreatment with ascorbate and JQ1 induced apoptosis and inhibited proliferation of cultured melanoma cells. Ascorbate deficiency as modeled in Gulo−/− mice diminished the treatment outcome of JQ1 for melanoma tumorgraft. In contrast, ascorbate supplementation lowered the effective dose of JQ1 needed to successfully inhibit melanoma tumors in mice. On the basis of our findings, future clinical trials with BETi should consider ascorbate levels in patients. Furthermore, ascorbate supplementation might help reduce the severe side effects that arise from BETi therapy by reducing the dosage necessary for treatment.Significance: This study shows that ascorbate can enhance the efficacy of BET inhibitors, providing a possible clinical solution to challenges arising in phase I trials from the dose-dependent side effects of this class of epigenetic therapy. Cancer Res; 78(2); 572–83. ©2017 AACR.

This file contains the following supplementary figures, tables and other materials. Figure S1: The expression of BRD2, BRD3 and BRD4 in melanocytes and melanoma cells. Figure S2: Knocking down the expression of BRD4 by siRNA. Figure S3: The effect of ascorbate on the expression of BRDs. Figure S4: hMeDIP-qPCR of HAT1 in its promoter and gene body. Figure S5: Decreased acetylation of soluble H4 by ascorbate treatment. Figure S6: The effect of ascorbate on H4K8ac and H4K16ac. Figure S7: Isobologram analysis of JQ1 and ascorbate interaction in A2058 cells. Figure S8: Ascorbate sensitizes murine melanoma cell B16-F10 to BETi. Table S1. Primers for hMeDIP-qPCR of HAT1. Table S2: Epigenetic drug screening panel. Table S3: Ascorbate decreases the EC50 of BET inhibitors in melanoma cell lines. Supplementary Material and Methods Supplementary reference

Supplementary Figure from DOT1L Is a Novel Cancer Stem Cell Target for Triple-Negative Breast Cancer

Supplementary Data from DOT1L Is a Novel Cancer Stem Cell Target for Triple-Negative Breast Cancer

Purpose:Although chemotherapies kill most cancer cells, stem cell–enriched survivors seed metastasis, particularly in triple-negative breast cancers (TNBC). TNBCs arise from and are enriched for tumor stem cells. Here, we tested if inhibition of DOT1L, an epigenetic regulator of normal tissue stem/progenitor populations, would target TNBC stem cells.Experimental Design:Effects of DOT1L inhibition by EPZ-5676 on stem cell properties were tested in three TNBC lines and four patient-derived xenograft (PDX) models and in isolated cancer stem cell (CSC)-enriched ALDH1+ and ALDH1− populations. RNA sequencing compared DOT1L regulated pathways in ALDH1+ and ALDH1− cells. To test if EPZ-5676 decreases CSC in vivo, limiting dilution assays of EPZ-5676/vehicle pretreated ALDH1+ and ALDH1− cells were performed. Tumor latency, growth, and metastasis were evaluated. Antitumor activity was also tested in TNBC PDX and PDX-derived organoids.Results:ALDH1+ TNBC cells exhibit higher DOT1L and H3K79me2 than ALDH1−. DOT1L maintains MYC expression and self-renewal in ALDH1+ cells. Global profiling revealed that DOT1L governs oxidative phosphorylation, cMyc targets, DNA damage response, and WNT activation in ALDH1+ but not in ALDH1− cells. EPZ-5676 reduced tumorspheres and ALDH1+ cells in vitro and decreased tumor-initiating stem cells and metastasis in xenografts generated from ALDH1+ but not ALDH1− populations in vivo. EPZ-5676 significantly reduced growth in vivo of one of two TNBC PDX tested and decreased clonogenic 3D growth of two other PDX-derived organoid cultures.Conclusions:DOT1L emerges as a key CSC regulator in TNBC. Present data support further clinical investigation of DOT1L inhibitors to target stem cell–enriched TNBC.

Replicative senescence limits the number of times primary cells can divide and is therefore regarded as a potential checkpoint for cancer progression. The majority of studies examining changes of gene expression upon senescence have been made with stationary senescent cells. We wanted to study the transition from normal growth to senescence in detail and identify early regulators of senescence by analyzing early changes in global gene expression, using Affymetrix microarrays. For this purpose, we used a murine epithelial senescence model, where senescence is abrogated by SV40 large T antigen and can be induced by using a temperature-sensitive form of SV40 large T antigen (SV40ts58). Comparisons were made to wild-type SV40 large T antigen-expressing cells and to cells expressing SV40ts58 large T antigen grown to confluence. After removal of genes that are similarly regulated in wild-type and temperature-sensitive SV40 large T antigen-expressing cells, 60% of the remaining genes were shared between cells arrested by inactivation of SV40 T antigen and by confluence. We identified 125 up-regulated and 39 down-regulated candidate genes/expressed sequence tags that are regulated upon SV40 T antigen inactivation and not during heat shock or confluence and classified these based on their kinetic profiles. Our study identified genes that fall into different functional clusters, such as transforming growth factor-β-related genes and transcription factors, and included genes not identified previously as senescence associated. The genes are candidates as early regulators of the senescence checkpoint and may be potential molecular targets for novel anticancer drugs.

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Purpose: Although chemotherapies kill most cancer cells, stem cell–enriched survivors seed metastasis, particularly in triple-negative breast cancers (TNBC). TNBCs arise from and are enriched for tumor stem cells. Here, we tested if inhibition of DOT1L, an epigenetic regulator of normal tissue stem/progenitor populations, would target TNBC stem cells. Experimental Design: Effects of DOT1L inhibition by EPZ-5676 on stem cell properties were tested in three TNBC lines and four patient-derived xenograft (PDX) models and in isolated cancer stem cell (CSC)-enriched ALDH1+ and ALDH1− populations. RNA sequencing compared DOT1L regulated pathways in ALDH1+ and ALDH1− cells. To test if EPZ-5676 decreases CSC in vivo, limiting dilution assays of EPZ-5676/vehicle pretreated ALDH1+ and ALDH1− cells were performed. Tumor latency, growth, and metastasis were evaluated. Antitumor activity was also tested in TNBC PDX and PDX-derived organoids. Results: ALDH1+ TNBC cells exhibit higher DOT1L and H3K79me2 than ALDH1−. DOT1L maintains MYC expression and self-renewal in ALDH1+ cells. Global profiling revealed that DOT1L governs oxidative phosphorylation, cMyc targets, DNA damage response, and WNT activation in ALDH1+ but not in ALDH1− cells. EPZ-5676 reduced tumorspheres and ALDH1+ cells in vitro and decreased tumor-initiating stem cells and metastasis in xenografts generated from ALDH1+ but not ALDH1− populations in vivo. EPZ-5676 significantly reduced growth in vivo of one of two TNBC PDX tested and decreased clonogenic 3D growth of two other PDX-derived organoid cultures. Conclusions: DOT1L emerges as a key CSC regulator in TNBC. Present data support further clinical investigation of DOT1L inhibitors to target stem cell–enriched TNBC.

Bromodomain and extraterminal inhibitors (BETi) are promising cancer therapies, yet prominent side effects of BETi at effective doses have been reported in phase I clinical trials. Here, we screened a panel of small molecules targeting epigenetic modulators against human metastatic melanoma cells. Cells were pretreated with or without ascorbate (vitamin C), which promotes DNA demethylation and subsequently changes the sensitivity to drugs. Top hits were structurally unrelated BETi, including JQ1, I-BET151, CPI-203, and BI-2536. Ascorbate enhanced the efficacy of BETi by decreasing acetylation of histone H4, but not H3, while exerting no effect on the expression of BRD proteins. Histone acetyltransferase 1 (HAT1), which catalyzes H4K5ac and H4K12ac, was downregulated by ascorbate mainly via the TET-mediated DNA hydroxymethylation pathway. Loss of H4ac, especially H4K5ac and H4K12ac, disrupted the interaction between BRD4 and H4 by which ascorbate and BETi blocked the binding of BRD4 to acetylated histones. Cotreatment with ascorbate and JQ1 induced apoptosis and inhibited proliferation of cultured melanoma cells. Ascorbate deficiency as modeled in Gulo−/− mice diminished the treatment outcome of JQ1 for melanoma tumorgraft. In contrast, ascorbate supplementation lowered the effective dose of JQ1 needed to successfully inhibit melanoma tumors in mice. On the basis of our findings, future clinical trials with BETi should consider ascorbate levels in patients. Furthermore, ascorbate supplementation might help reduce the severe side effects that arise from BETi therapy by reducing the dosage necessary for treatment. Significance: This study shows that ascorbate can enhance the efficacy of BET inhibitors, providing a possible clinical solution to challenges arising in phase I trials from the dose-dependent side effects of this class of epigenetic therapy. Cancer Res; 78(2); 572–83. ©2017 AACR.

This abstract was withdrawn by the authors.

Replicative senescence limits the number of times primary cells can divide and is therefore regarded as a potential checkpoint for cancer progression. The majority of studies examining changes of gene expression upon senescence have been made with stationary senescent cells. We wanted to study the transition from normal growth to senescence in detail and identify early regulators of senescence by analyzing early changes in global gene expression, using Affymetrix microarrays. For this purpose, we used a murine epithelial senescence model, where senescence is abrogated by SV40 large T antigen and can be induced by using a temperature-sensitive form of SV40 large T antigen (SV40ts58). Comparisons were made to wild-type SV40 large T antigen-expressing cells and to cells expressing SV40ts58 large T antigen grown to confluence. After removal of genes that are similarly regulated in wild-type and temperature-sensitive SV40 large T antigen-expressing cells, 60% of the remaining genes were shared between cells arrested by inactivation of SV40 T antigen and by confluence. We identified 125 up-regulated and 39 down-regulated candidate genes/expressed sequence tags that are regulated upon SV40 T antigen inactivation and not during heat shock or confluence and classified these based on their kinetic profiles. Our study identified genes that fall into different functional clusters, such as transforming growth factor-β-related genes and transcription factors, and included genes not identified previously as senescence associated. The genes are candidates as early regulators of the senescence checkpoint and may be potential molecular targets for novel anticancer drugs.

Bromodomain and extra-terminal domain (BET) proteins have emerged as promising therapeutic targets in glioblastoma and many other cancers. Small molecule mimics of acetylated histones inhibit BET protein function and reduce expression of several oncogenes required for glioblastoma (GBM) progression. However, the mechanism through which BET protein inhibition reduces GBM growth is not completely understood. Long noncoding RNAs (lncRNAs) are important epigenetic regulators with critical roles in cancer initiation and malignant progression but mechanistic insight into their expression and regulation by bromodomain inhibitors remains elusive. In this study we utilized single molecule sequencing (SMS) to comprehensively profile lncRNAs differentially expressed in GBM and we identified a subset of GBM-specific lncRNAs whose expression is regulated by BET proteins. Treatment of GBM cells with the BET protein inhibitor I-BET151 reduced levels of the tumor-promoting lncRNA HOTAIR and restored the expression of several other GBM-down-regulated lncRNAs. Conversely, overexpression of HOTAIR in conjunction with I-BET treatment rescues the antiproliferative activity of I-BET151. Moreover, chromatin immunoprecipitation analysis demonstrated binding of Brd4 to the HOTAIR promoter suggesting that BET proteins can directly regulate lncRNA expression. Our data unravel a previously unappreciated mechanism through which BET proteins control tumor growth of glioblastoma cells and suggest that lncRNA networks may, in part, mediate the anti-proliferative effects of many epigenetic inhibitors currently in clinical trials for cancer and other diseases. Note: This abstract was not presented at the meeting. Citation Format: Chiara Pastori, Philip Kapranov, Clara Penas, Georges St.Laurent,Nagi Ayad, Claes Wahlestedt. The Bromodomain inhibitors regulate long noncoding RNAs controlling glioblastoma progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4764. doi:10.1158/1538-7445.AM2015-4764

Triple-negative breast cancers (TNBC) is the aggressive subtype with limited treatment options and very poor prognosis following progression after standard chemotherapy regimens. There is an urgent clinical need to identify new therapeutic targets in order to improve the outcome for these patients. Increasing evidence suggest that cancer stem cells (CSCs) mediate therapy resistance and metastasis and may thus be responsible for cancer relapse and deaths in breast cancer patients. We have previously identified distinct subsets of CSCs in the most deadly form of TNBC cell lines and patient derived cultures. Within CD44+ populations, CD24 expression defines two subsets of CSC subpopulations: CD24neg and CD24+. Both CD44+CD24neg (CD24neg) and CD44+CD24+ (CD24+) populations have self-renewing and tumor-initiating properties, however, the CD24+ population has Notch1 activation, generates more spheres and colonies, and contains ALDH1+ and ESA+ subpopulations. CD24+ cells are also more efficient at producing xenograft tumors than CD24neg. Most importantly, only the CD24+ population can spontaneously metastasize from an orthotopic tumor xenograft. CD24+ cells can self-renew and give rise to CD24neg cells, while CD24neg progeny generate only CD24neg. Thus, identifying drugs that would eradicate the CD24+ population would of great clinical significance in the treatment of this deadly type of cancer. Here, we test the differential response of these subsets to chemotherapy and Gamma-Secretase Inhibitors (GSI). Surviving CD24+ cells were enriched by paclitaxel treatment: >80% of cells surviving at day 8 were CD24+ and survival of CD24neg progeny from CD24+ was dramatically attenuated. In contrast, the two TNBC subsets differed notably in their responses to RO4929097, a GSI in clinical trials for cancer. RO4929097 reduced the self-renewal of CD24+ cells, but had no effect on proliferation or survival of CD24neg. Only CD24+-generated tumors responded to RO4929097 while the drug had no effect on tumors derived from CD24neg cells, which comprise a majority of the population in TNBC lines. This data provides rationale for further testing of combined chemotherapy and Notch-targeting drugs in the treatment of this deadly cancer. It also supports the use of CD24 as a marker in TNBCs to screen and identify new therapeutic drugs that selectively target the most aggressive CSCs. Surface CD24 expression may also be used to further study the role of niche-induced interconversion between CSC subsets. Notch1-intracellular domain (NI-ICD) overexpression in CD24neg and CD24+ cells not only increased CD24+ cells indicating increased self-renewal, but also led to conversion of CD24neg cell to CD24+, an event we have not observed spontaneously in culture over several years. Taken together, these observations support the intriguing possibility that the microenvironment may regulate the phenotypic and functional heterogeneity present within CSCs. Citation Format: Diana Azzam, Shaun Brothers, Claes Wahlestedt, Joyce Slingerland. Therapeutic targeting of distinct subsets of cancer stem cells within triple negative breast cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3042. doi:10.1158/1538-7445.AM2014-3042

Glioblastoma (GBM) is the most common and aggressive primary brain malignancy in adults. The standard treatment consists of surgery followed by radiotherapy and chemotherapy, although this only modestly affects patient survival. Partial tumor resection and poor drug delivery to the brain impede effective treatment options. The development of high-throughput sequencing technology has allowed the cancer research community to identify hundreds of genetic alterations such as amplification, deletion, mutation, and changes in gene expression. However, all this information still needs to be translated into new therapeutic approaches. These novel therapies may come from understanding long non-coding RNAs (lncRNAs). lncRNAs modulate transcription, regulate post-transcriptional RNA processing, translation, DNA methylation, and chromatin architecture through local (cis) and long distance (trans) mechanisms. Although relatively few lncRNAs have been functionally characterized, fewer are reported to have oncogenic or tumor suppressor properties. Recently, small signatures of 6 long-noncoding RNAs with altered expression have been reported for glioblastoma and these lncRNAs have shown correlation to overall survival. Using the Helicos Next Generation Sequencing platform 12 GBM samples and 8 controls were sequenced. Our lab identified hundreds of lncRNAs potentially dysregulated in GBM. Among these there was the well-known lncRNA HOTAIR, which was found to be massively upregulated in GBM. This observation correlated with what has been observed in other cancers. However, increased expression of HOTAIR has been linked to poor prognosis due to metastatic events. Here we show that in glioblastoma HOTAIR does not promote metastasis, but instead sustains the ability of these cells to proliferate. We demonstrate that HOTAIR knockdown in GBM strongly impairs the proliferation of cells and induces apoptosis in vitro and in vivo experiments. To identify the direct gene targets of HOTAIR we employed the chirp technique (chromatin isolation by RNA precipitation), which allowed us to explain how this lncRNA affects gene transcription involved in proliferation/apoptosis and shed light on the cellular pathways regulated by HOTAIR. In addition to cell cycle regulation mediated by HOTAIR we find that certain epigenetic enzyme inhibitors potently downregulate HOTAIR expression. These exciting studies define a novel mechanism through which these enzymes control cell proliferation via lncRNA expression. Citation Format: Chiara Pastori, Clara Penas, Philipp Kapranov, Georges St.Laurent, Ming Zhang, Nagi Ayad, Claes Wahlestedt. The long noncoding RNA HOTAIR promotes glioblastoma cell proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-114. doi:10.1158/1538-7445.AM2014-LB-114