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Tumor cell resistance to ferroptosis, which is a recently discovered CTL induced cell death pathway, remains incompletely understood. We report here that interferon regulatory factor 8 (IRF8) functions as a regulator of tumor cell intrinsic ferroptosis. Genome-wide gene expression profiling identified ferroptosis pathway as an IRF8-regulated pathway in tumor cells. IRF8.KO tumor cells are resistant to intrinsic ferroptosis induction and also exhibit decreased ferroptosis in response to tumor specific CTL killing. Loss of IRF8 increases p53 express in tumor cells and knocking out Tp53 in IRF8.KO cells restored tumor cell sensitivity to intrinsic ferroptosis induction. We were able to show that treating tumor-bearing mice with IRF8-encoding plasmid NTC9385R-mIRF8 encapsulated in a DOTAP-cholesterol lipid nanoparticle significantly reduced tumor growth and increased the percentage of dead cells in the tumor. Our data therefore determine that IRF8 represses p53 expression to maintain tumor cell sensitivity to intrinsic ferroptosis. Citation Format: Dakota Poschel, Mercy Kehinde-Ige, John Klement, Dafeng Yang, Alyssa Merting, Natasha Savage, Huidong Shi, Kebin Liu. IRF8 regulates tumor cell sensitivity to intrinsic ferroptosis by repressing p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB036.

Adoptive cell therapy has revolutionized the landscape of cancer immunotherapy, exhibiting unprecedented success in treating various types of cancer. Substantial investigative efforts have been devoted to gaining a deeper understanding of the ways in which adoptively transferred T cells eliminate tumors and to discovering strategies that enhance T cell functionality in the tumor microenvironment. We have established an experimental mouse model in which adoptive transfer of tumor-specific CD4+ T cells leads to eradication of established tumors. Using this model, we previously reported on the profound disruption of tumor metabolism induced by adoptive T cell therapy. However, the detailed mechanisms that enable tumor-reactive CD4+ T cells to alter tumor metabolism remain largely undefined. In the current study, we aimed to identify the key metabolic enzymes and pathways targeted by antitumor CD4+ T cells that lead to progressive tumor regression. Utilizing next-generation sequencing strategies, including bulk and single-cell RNA sequencing, we examined the metabolic changes cancer cells undergo in response to treatment, and the immune cell compositions in the tumor microenvironment. Additionally, we performed an in vivo CRISPR knockout screen to identify the metabolic genes that either sensitize tumor cells to adoptive cell therapy or contribute to tumor resistance. Results from our study may also illuminate potential targets in cancer metabolism that can be exploited using small-molecule inhibitors when adoptive cell therapy is not feasible. Citation Format: Caitlin Brandle, Ogacheko D. Okoko, Md Yeashin Gazi, Xin Wang, Mercy Kehinde-Ige, Huidong Shi, Gang Zhou. Antitumor CD4+ T cells reprogram tumor metabolism to drive progressive tumor regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 268.

Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNA-Seq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. We performed scRNA-Seq and CyTOF analyses of the myeloid cell populations of tumors and spleens from metastatic 4T1 and non-invasive EMT6 tumor-bearing mice. Tumors and spleens from 4T1 tumor-bearing mice exhibited a marked expansion of myeloid cell subsets that are characterized by the expression of immunosuppressive as well as progenitor markers. On the contrary, indicated tissues from EMT6 mice were enriched in NK cells, T and B lymphocytes and they were lacking immunosuppressive myeloid cell subsets. Furthermore, we identified a distinct differentiation pattern of immature myeloid cell subsets from neutrophil progenitors (NP) in 4T1 tumor-bearing mice. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Hasan Korkaya, Elayne Benson, Fulya Koksalar Alkan, Justin Wilson, Tulshi Patel, Hilmi K. Alkan, Virginia McEvoy, Nika Shekastehband, Nate Francois, Huidong Shi, Catherine C. Hedrick. Phenotypic & functional diversity of tumor associated neutrophils in murine breast tumor models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4600.

Adoptive Cell Therapy (ACT) holds great promise to transform cancer treatments. However, applying ACT to a broad patient population still faces major challenges. The significant barriers to the success of ACT have been attributed to multiple factors, including functional exhaustion, poor persistence, and the inability of T cells to efficiently compete against tumor cells for metabolic resources in the tumor microenvironment (TME). These functional deficiencies highlight the importance of targeting T cell metabolism to improve ACT treatment outcomes. In the TME, tumor cells preferentially utilize glycolysis to rapidly generate ATP, converting pyruvate into lactate and creating a hypoxic environment that is inconducive for effector T cells due to a lack of access to the metabolites needed for sustenance, leading to T cell exhaustion, and declined proliferation. Consequently, T cells lose their function and are unable to persist against tumor cells. The expression of Constitutively Active Signal Transducer and Activator of Transcription 5A (CASTAT5) on tumor-specific CD4+ T cells has been shown to improve their persistence, polyfunctionality, and anti-tumor effects. Our hypothesis is that the improved polyfunctionality and persistence of CASTAT5-CD4+ T cells are partly due to the reprogramming of their metabolic landscape by CASTAT5, resulting in improved metabolic fitness. Single-cell metabolic pathway enrichment analysis of the identified polyfunctional CASTAT5-CD4+ T cells revealed increased capacity for glycolysis, electron transport chain, oxidative phosphorylation, and amino acid metabolism. Single-cell Flux Estimation Analysis (scFEA) further revealed increased metabolic fluxes in amino acid uptake and synthesis, glycolysis, tricarboxylic acid (TCA) cycle, and polyamine synthesis. Interestingly, CASTAT5-CD4+ T cells showed increased polyamine (spermine and spermidine) synthesis, with the upregulation of the metabolic fluxes from putrescine to spermine, catalyzed by spermidine synthase (Srm) and spermine synthase (Srs), both of which are also upregulated in the polyfunctional CD4+ T cell populations. Spermidine and spermine have been shown to enhance mitochondrial metabolism in T cells, increasing their proliferation and survival. Here, we show that there is increased synthesis of spermine in CASTAT5-CD4+ T cells, which may contribute to their increased capacity for oxidative phosphorylation and TCA cycle, leading to the observed improvements in their persistence and anti-tumor function. These findings suggest a role for the polyamine synthesis pathway in the improvement of T cell function. Further studies modulating targets within the polyamine synthesis pathway will help provide an improved understanding of the underlying mechanisms affecting T cell function, thus, unveiling potential metabolic targets to improve the efficacy of tumor-specific T cells. Citation Format: Mercy Kehinde-Ige, Ogacheko Okoko, Zhi-Chun Ding, Gang Zhou, Huidong Shi. Single-cell flux estimation analysis reveals the metabolic states of tumor-specific CD4+ T cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3693.

Although accumulation of myeloid-derived suppressor cells (MDSC) is a hallmark of cancer, the underlying mechanism of this accumulation within the tumor microenvironment remains incompletely understood. We report here that TNFα–RIP1–mediated necroptosis regulates accumulation of MDSCs. In tumor-bearing mice, pharmacologic inhibition of DNMT with the DNA methyltransferease inhibitor decitabine (DAC) decreased MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes. DAC-induced decreases in MDSC accumulation correlated with increased expression of the myeloid cell lineage-specific transcription factor IRF8 in MDSCs. However, DAC also suppressed MDSC-like cell accumulation in IRF8-deficient mice, indicating that DNA methylation may regulate MDSC survival through an IRF8-independent mechanism. Instead, DAC decreased MDSC accumulation by increasing cell death via disrupting DNA methylation of RIP1-dependent targets of necroptosis. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter was hypermethylated in tumor-induced MDSCs in vivo. DAC treatment dramatically increased TNFα levels in MDSC in vitro, and neutralizing TNFα significantly increased MDSC accumulation and tumor growth in tumor-bearing mice in vivo. Recombinant TNFα induced MDSC cell death in a dose- and RIP1-dependent manner. IL6 was abundantly expressed in MDSCs in tumor-bearing mice and patients with human colorectal cancer. In vitro, IL6 treatment of MDSC-like cells activated STAT3, increased expression of DNMT1 and DNMT3b, and enhanced survival. Overall, our findings reveal that MDSCs establish a STAT3–DNMT epigenetic axis, regulated by autocrine IL6, to silence TNFα expression. This results in decreased TNFα-induced and RIP1-dependent necroptosis to sustain survival and accumulation. Significance: These findings demonstrate that targeting IL6 expression or function represent potentially effective approaches to suppress MDSC survival and accumulation in the tumor microenvironment.

Despite the advances in early diagnostics and therapeutics, women with metastatic breast cancer have limited treatment options. Women with TNBC, who constitute 15-20% of breast cancer patients, are often diagnosed with aggressive/metastatic disease. Advanced studies implicated immunosuppressive tumor microenvironment (TME) in aggressive/metastatic properties of TNBC subtype. Alternatively activated immature myeloid cells including tumor-associated macrophages (TAM), tumor-associated neutrophils (TAN), tumor-associated dendritic cells (TADC) and myeloid derived suppressor cells (MDSC) constitute a major component of TME. However, anti-tumorigenic microenvironment is also reported and that may have clinical relevance in early TNBC patients. Therefore, our hypothesis is that myeloid cells polarize to become immunosuppressive and infiltrate tumors and pre-metastatic niches in patients with advanced disease, while patients with early TNBCs may elicit anti-tumor immune response eliminating disseminated tumor cells (DTC). The utilization of syngeneic immunocompetent mouse models has contributed to our current understanding of immunosuppressive or immunomodulatory TME. Using these models, we have demonstrated that tumor dissemination and growth at metastatic sites is facilitated by MDSC’s. Emerging technologies; single cell RNA sequencing (scRNAseq), mass cytometry (CyTOF) or cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) has been powerful platforms for detailed characterization of tumors and TME compartments. Our bulk gene expression data of the myeloid cell populations of tumor microenvironment, lung, spleen and BM from 4T1 tumor-bearing mice showed distinct MDSC gene signatures. When applied to publicly available scRNAseq data, lung gMDSCs from 4T1 metastatic tumor model appeared to show different trajectory of polarization than the tumor gMDSCs. Consistent with previous findings by Hedrick Lab, lung gMDSCs from 4T1 mice also express higher levels of NeP markers compared to BM and tumor gMDSCs as well as lung gMDSCs from EMT6 mice. However, analyses of immune cells from EMT6 tumor bearing mice exhibited an anti-tumor immune signature which is consistent with the clearance of the DTCs following complete resection of the primary tumors. Using the murine TNBC models in syngeneic mice, we provide evidence that early TNBC tumors may elicit anti-tumor immune responses and thus the survival outcome in those patients is substantially increased after complete surgical resection of the primary tumors. Whereas immunosuppressive tumor microenvironment contributes to the poor overall survival in patients with advanced TNBCs. Therefore, identifying an anti-tumor immune signature in early TNBC patients may be utilized as a clinical biomarker before surgical intervention as well as improve the survival outcome. Citation Format: Fulya Alkan, Raziye Piranlioglu, Eunmi Lee, Maria Ouzounova, Catherine C Hedrick, Huidong Shi, Hasan Korkaya. Understanding the pro- and anti-tumorigenic microenvironments in syngeneic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2223.

Splicing factor SF3B1 is frequently mutated in chronic lymphocytic leukemia (CLL) patients and has been suggested as a potential therapeutic target. In this study, we demonstrated that SF3B1 modulator sudemycin D6 (SD6) effectively inhibits cell growth and induces apoptosis in CLL cells. RNA sequencing analysis revealed significant increases in global intron retention in SD6-treated CLL cells. Pathway analysis of the genes associated with increased intron retention suggested that B-cell receptor (BCR) and PI3K signaling pathways were among the most important pathways being affected by SD6. The increases in intron retention were inversely correlated with decreases in mRNA and protein levels of the affected BCR/PI3K pathway molecules including BLNK, BTK, AKT, PLCγ2, and PI3Kδ. SD6 treatment also induced a time-dependent exon-skipping event in MCL-1 mRNA and resulted in significant down-regulation of another anti-apoptotic gene TRAF1, thus collectively contributing to the SD6-induced apoptosis. Furthermore, SD6 treatment can overcome the pro-survival and pro-growth signals and synergize with established CLL therapies ibrutinib, idelalisib, and venetoclax to induce apoptosis in primary CLL cells co-cultured with bone marrow stromal cells and T-cell-derived cytokines. Finally, in vivo treatment with SD6 at 10mg/kg/day for 7 days significantly inhibited the growth of xenograft tumors that were established by subcutaneous inoculation of 5×106 MEC1 CLL cells into NOD mice. Collectively, these results provide a strong rationale for the future clinical development of spliceosome modulators and potential combination therapies for the treatment of CLL. Citation Format: Qimei Han, Jianbo Wang, Austin Y. Shull, Fang Shi, Libin Deng, Jeong-Hyeon Choi, Eun-Jeong Park, Shuo Tu, Lirong Pei, Farrukh T. Awan, Roni Bollag, Locke J. Bryan, Hong-bo Xin, Chandraiah Lagisetti, Thomas R. Webb, Victor Jin, Huidong Shi. Modulation of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5230.

SF3B1 mutation, which occurs in 10-20% of chronic lymphocytic leukemia (CLL) patients, is associated with faster disease progression, shorter overall survival and fludarabine resistance. Mutant SF3B1 utilizes cryptic 3′ splice sites to generate aberrantly spliced mRNAs, half of which may undergo non-sense mediated mRNA decay (NMD), leading to downregulation of the affected genes. To identify potential tumor suppressor genes that are aberrantly spliced and downregulated by mutant SF3B1, we performed RNA-sequencing analysis of SF3B1-mutated primary CLL cells in the presence or absence of cycloheximide (CHX), a translation inhibitor known to inhibit NMD. Our analysis identified PPP2R5A, encoding one of the regulatory subunits of protein phosphatase-2A (PP2A B56α), as one of the key genes that were most consistently affected by an aberrantly spliced junction, and significantly downregulated (>2 fold) in CLL patient samples with various SF3B1 hotspot mutations. Splicing analyses by RT-PCR and Sanger sequencing confirmed that a 13-nucleotides sequence was added before the 5th exon of PPP2R5A via alternative splicing in CLL patients with the SF3B1 mutation. Due to this 13-nucleotides addition, three consecutive premature stop codons were created by frameshift at a position more than 55 bases upstream of an exon-exon junction, which is a canonical feature of the mRNAs degraded through NMD. The down-regulation of PPP2R5A was confirmed by quantitative PCR (p Citation Format: Qimei Han, Libin Deng, Shuo Tu, Lirong Pei, Jeong-Hyeon Choi, Victor Jin, Huidong Shi. SF3B1 mutation promotes c-Myc protein stability through aberrant splicing and downregulation of PP2A B56α subunit in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4493.

CD73 is the major extracellular adenosine-generating ecto-5'-nucleotidase (Nt5e) that metabolizes extracellular (e) AMP and generates immunosuppressive adenosine (eADO). Elevated CD73 expression is often exploited by tumors and other cellular constituents of the tumor microenvironment (TME) as a mechanism of immune evasion. Clinically, a high level of CD73 expression in the tumor microenvironment (TME) is linked to poor prognosis. Experimental evidence also demonstrates that host CD73 activity in both hematopoietic and non-hematopoietic compartments augments the suppressive milieu of the TME. Nevertheless, the identities of CD73-expressing non-hematopoietic cells and factors that regulate CD73 activity of these cells have yet to be clearly defined. Our recent results unravel that cancer associated fibroblasts (CAFs) constitute a crucial non-hematopoietic CD73hi compartment in the TME of human colorectal cancer (CRC) and murine MC38 CRC and EG7 lymphoma ectopic models. Strikingly, the CD73 expression profile among cohorts of published human CRC and breast cancer patient data sets confirmed its significant positive association with that of CAF-related markers, both of which are negatively correlated with poor survival. Cellular and transcriptomic analyses of CAFs in the EG7 TME reveal that CD73 expression in CAFs increases progressively during tumor progression, associated with elevated tissue hypoxia. Mechanistically, we demonstrate that the progressive elevation of CD73 expression in CAFs during tumor progression is caused by elevated eADO in the TME, which is resulted by the hypoxia/necrosis-associated eATP release. Furthermore, this eADO/adenosingeric pathway-induced CD73 upregulation in CAFs is mostly mediated by activation of A2BR and downstream MAPK signaling, which exacerbates eADO accumulation and serves as a feedforward loop of re-enforcing the immunosuppressive TME. Our study demonstrated that clinically and experimentally, high level of CD73 expression in CAFs represents another crucial immune checkpoint in the TME. Targeted inactivation or blockade of A2BR signaling may serve as a new strategy to prevent or alleviate the initiation of the eADO-CD73-associated amplification of immunosuppression in the TME, thereby improving the outcome of immunotherapy. Citation Format: Miao Yu, Gang Guo, Lei Huang, Libin Deng, Roni Bollag, Andrew Mellor, Huidong Shi, Yan Cui. Dynamic regulation of CD73 expression in cancer associated fibroblasts enforces immunosuppression via a feedforward adenosinergic pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-282.

We developed a novel approach for conducting multisample, multigene, ultradeep bisulfite sequencing analysis of DNA methylation patterns in clinical samples. A massively parallel sequencing-by-synthesis method (454 sequencing) was used to directly sequence >100 bisulfite PCR products in a single sequencing run without subcloning. We showed the utility, robustness, and superiority of this approach by analyzing methylation in 25 gene-related CpG rich regions from >40 cases of primary cells, including normal peripheral blood lymphocytes, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). A total of 294,631 sequences was generated with an average read length of 131 bp. On average, >1,600 individual sequences were generated for each PCR amplicon far beyond the few clones (

Recently, impressive technical advancements have been made in the isolation and validation of mammary stem cells and cancer stem cells (CSC), but the signaling pathways that regulate stem cell self-renewal are largely unknown. Furthermore, CSCs are believed to contribute to chemo- and radioresistance. In this study, we used the MMTV-Neu-Tg mouse mammary tumor model to identify potential new strategies for eliminating CSCs. We found that both luminal progenitor and basal stem cells are susceptible to genetic and epigenetic modifications, which facilitate oncogenic transformation and tumorigenic potential. A combination of the DNMT inhibitor 5-azacytidine and the HDAC inhibitor butyrate markedly reduced CSC abundance and increased the overall survival in this mouse model. RNA-seq analysis of CSCs treated with 5-azacytidine plus butyrate provided evidence that inhibition of chromatin modifiers blocks growth-promoting signaling molecules such as RAD51AP1 and SPC25, which play key roles in DNA damage repair and kinetochore assembly. Moreover, RAD51AP1 and SPC25 were significantly overexpressed in human breast tumor tissues and were associated with reduced overall patient survival. In conclusion, our studies suggest that breast CSCs are intrinsically sensitive to genetic and epigenetic modifications and can therefore be significantly affected by epigenetic-based therapies, warranting further investigation of combined DNMT and HDAC inhibition in refractory or drug-resistant breast cancer. Cancer Res; 76(11); 3224–35. ©2016 AACR.

Tumor infiltrating lymphocytes (TILs) play a critical role in regulating the immunomodulatory properties of triple negative breast cancer (TNBC). However, the specific adaptations that TNBC tumors undergo when challenged by lymphocyte infiltration remain unclear. In order to address this gap in knowledge, we conducted an immuno-phenotype comparison using mRNA sequencing between the TNBC cell line MDA-MB-231 and the luminal breast cancer cell line MCF7 after both were co-cultured with activated human T-cells. Although the cytokine-induced immune signature of the two cell lines were similar, MDA-MD-231 cells were able to transcribe the tryptophan catabolizing enzyme IDO1 at a significantly higher level than MCF7 cells. Stimulation with IFNg was able to differentially induce IDO protein expression and enzymatic activity in ER- cell lines compared to ER+ cell lines, though no differences were observed in upstream JAK/STAT1 signaling or IDO1 mRNA stability between the two cell lines. Further experiments showed that treatment with the demethylating agent 5-aza-deoxycytidine was able to reverse suppression of IDO1 expression in MCF7 cells, suggesting that DNA methylation serves as a potential determinant in IDO1 induction. Analysis of TCGA and other previously published breast cancer datasets revealed subtype-specific mRNA and promoter methylation differences in IDO1, with TNBC/basal-like subtypes exhibiting lower promoter methylation and higher mRNA expression than ER+/luminal subtypes. Bisulfite pyrosequencing validated the subtype-specific association of decreased promoter methylation with increased IDO1 expression in breast cancer cell lines and an independent cohort of primary breast tumors. In addition, decreased IDO1 promoter methylation and elevated IDO1 expression in basal-like breast tumors was found to be associated with increased levels of kynurenine, the metabolic product of IDO1, as well as higher numbers of CD8+ TILs. Furthermore, high kynurenine levels in breast tumors were associated with worse patient survival. Taken together, these findings suggest that subtype-specific IDO1 promoter methylation regulates the ability of breast tumors to escape from antitumor immune responses driven by CD8+ TILs and could be used as a predictive biomarker for IDO inhibitor-based immunotherapy. Citation Format: Gu F, Noonepalle SK, Lee E-J, Choi J-H, Shull AY, Pei L, Sreekumar A, Ambs S, Shi H. Modulation of indoleamine 2, 3-dioxygenase (IDO1) expression in breast cancer cells by activated CD8+ T cells is controlled by DNA promoter methylation [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-02-08.

Chronic lymphocytic leukemia (CLL) is a malignancy characterized by the progressive accumulation of CD19+ B-cells capable of overcoming their regulated life cycle. Because this disease is highly heterogeneous and utilizes several mechanisms to resist apoptotic death, CLL currently remains incurable. Based on the ongoing efforts to interpret the transcriptional dynamics of CLL pathogenicity as well as prior reports demonstrating the efficacy of cyclin-dependent kinase (CDK) transcriptional inhibitors, we sought to characterize the molecular response of CLL cells treated with the covalent CDK7 inhibitor THZ1 and determine the therapeutic potential of CDK7 inhibition in CLL. We first observed that THZ1 was able to inhibit growth in malignant B-cell lines MEC1 and MEC2 in both a dose dependent (IC50: .045uM & .030uM, respectively) and time dependent manner. The inhibition in cell growth corresponded with both G1-mediated cell cycle arrest as well as apoptosis in the respective cell lines. We also observed dose-dependent reduction in cell viability through apoptosis in patient-derived CLL B-cells after 24 hours. Because of the observed outcomes of THZ1-treated CLL cells, we then wanted to determine the specific transcriptional targets significantly suppressed by THZ1 treatment. To identify the THZ1-sensitive transcripts, we performed a time course RNAseq expression analysis in both MEC1 and MEC2 treated with 50nM THZ1. Based on the incremental reduction of covered reads over a 12-hour period, we determined that the top 50 transcripts greatly diminished in both MEC1 and MEC2 contain significant enrichment in genes attributed to glycolytic metabolism (ex: ALDOC, SLC2A1, TPI1, GAPDH, ENO2). Along with the overlapping comparison between the two cell lines, we next compared the downregulated transcripts from the treated cell lines against the RNAseq expression profile of 47 CLL patients and 5 healthy donors. We observed that THZ1 was able to suppress transcripts upregulated in CLL patient samples including ENO2, FGR, WNT10A, and CBX7 in MEC1 and ENO2, MALAT1, CCR7, and PLCG1 in MEC2. Finally, we performed H3K27Ac ChIPseq in MEC1 to identify super enhancers that may overlap with THZ1-sensitive transcripts. From this comparison, we determined that super enhancers exist within reported oncogenic drivers CBX7, WNT10A, and FGR. Overall, we observe that THZ1 can effectively overcome the anti-apoptotic phenotype of CLL B-cells as well as directly suppress transcription of genes that can drive CLL malignancy. With the addition of CDK7 ChIPseq, our ultimate goal is to provide clarity regarding CDK7-mediated transcriptional regulation in CLL and demonstrate the molecular consequences of therapeutic CDK7 inhibition in CLL. Citation Format: Austin Young Shull, Jeong-Hyeon Choi, Jordan Bauer, Lirong Pei, Farrukh T. Awan, Huidong Shi. The covalent CDK7 inhibitor THZ1 can counteract apoptotic resistance and suppress the transcription of genes attributed to chronic lymphocytic leukemia malignancy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4697.

Splicing factor SF3B1 is frequently mutated in about 10-15% of CLL patients. SF3B1 mutations are predominately associated with faster disease progression and poor overall survival, and mutations are typically enriched in approximately 20% relapsed and chemorefractory CLL patients. Therefore, there is a growing interest in using SF3B1 as a therapeutic target for CLL. In this study, we investigated the effectiveness of Sudemycin D6 (SD6), a small molecule SF3B1 inhibitor, in reducing cell viability and promoting apoptosis in CLL cells. We then isolated RNA from both the SD6-treated and control cells at 2, 6, and 24hr post-treatment and performed RNA sequencing analysis to investigate the global impact. We demonstrated that SD6 effectively inhibits cell growth and induces apoptosis in CLL cells at a nanomolar concentration in both a time and dosage dependent manner. Furthermore, global analysis of the RNA sequencing data revealed that treatment with SD6 causes an aberrant splicing signature with significant increase in global intron retention. The increase in global intron retention peaked at 6hr then significantly diminished at 24hr post-treatment. Ontology analysis of the intron-retaining transcripts using the Ingenuity Pathway Analysis (IPA) software package suggested that B-cell receptor (BCR) signaling, protein ubiquitination, and PI3K signaling were among the top canonical pathways affected by SD6 treatment. Using RT-PCR analysis, we confirmed intron retention events in several components of the BCR pathway, such as BTK, BLNK, and PIK3CD, and an exon-skipping event in the exons encoding the kinase domain of Bruton's Tyrosine Kinase. The increase in intron retention significantly correlated with a decrease in overall mRNA and protein expression levels of the affected downstream BCR pathway proteins like AKT, PI3Kδ, and PLCγ2. Furthermore, SD6 treatment induced a time-dependent exon-skipping event in the apoptotic regulator MCL1, resulting in alternative splicing of the long, anti-apoptotic isoform of MCL1 into the short, pro-apoptotic isoform. SD6 treatment also caused downregulation of anti-apoptotic gene TRAF1 at both the mRNA and protein level, which could contribute to SD6-induced apoptosis. Cumulatively, these results strongly suggest that SF3B1 is a promising target for therapeutic treatment in CLL. Our study also provides solid rationale for future clinical development of spliceosome inhibitors and combination therapies based on spliceosome inhibitors. Citation Format: Qimei Han, Austin Young Shull, Fang Shi, Chandraiah Lagisetti, Thomas Webb, Justin Choi, Huidong Shi. Inhibition of SF3B1 causes global intron retention and downregulation of the B-cell receptor pathway in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1997.

Although alterations in xenobiotic metabolism are considered causal in the development of bladder cancer, the precise mechanisms involved are poorly understood. In this study, we used high-throughput mass spectrometry to measure over 2,000 compounds in 58 clinical specimens, identifying 35 metabolites which exhibited significant changes in bladder cancer. This metabolic signature distinguished both normal and benign bladder from bladder cancer. Exploratory analyses of this metabolomic signature in urine showed promise in distinguishing bladder cancer from controls and also nonmuscle from muscle-invasive bladder cancer. Subsequent enrichment-based bioprocess mapping revealed alterations in phase I/II metabolism and suggested a possible role for DNA methylation in perturbing xenobiotic metabolism in bladder cancer. In particular, we validated tumor-associated hypermethylation in the cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1 (CYP1B1) promoters of bladder cancer tissues by bisulfite sequence analysis and methylation-specific PCR and also by in vitro treatment of T-24 bladder cancer cell line with the DNA demethylating agent 5-aza-2′-deoxycytidine. Furthermore, we showed that expression of CYP1A1 and CYP1B1 was reduced significantly in an independent cohort of bladder cancer specimens compared with matched benign adjacent tissues. In summary, our findings identified candidate diagnostic and prognostic markers and highlighted mechanisms associated with the silencing of xenobiotic metabolism. The metabolomic signature we describe offers potential as a urinary biomarker for early detection and staging of bladder cancer, highlighting the utility of evaluating metabolomic profiles of cancer to gain insights into bioprocesses perturbed during tumor development and progression. Cancer Res; 71(24); 7376–86. ©2011 AACR.

This study examined DNA methylation associated with acute lymphoblastic leukemia (ALL) and showed that selected molecular targets can be pharmacologically modulated to reverse gene silencing. A CpG island (CGI) microarray containing more than 3,400 unique clones that span all human chromosomes was used for large-scale discovery experiments and led to 262 unique CGI loci being statistically identified as methylated in ALL lymphoblasts. The methylation status of 10 clones encompassing 11 genes (DCC, DLC-1, DDX51, KCNK2, LRP1B, NKX6-1, NOPE, PCDHGA12, RPIB9, ABCB1, and SLC2A14) identified as differentially methylated between ALL patients and controls was independently verified. Consequently, the methylation status of DDX51 was found to differentiate patients with B- and T-ALL subtypes (P = 0.011, Fisher's exact test). Next, the relationship between methylation and expression of these genes was examined in ALL cell lines (NALM-6 and Jurkat) before and after treatments with 5-aza-2-deoxycytidine and trichostatin A. More than a 10-fold increase in mRNA expression was observed for two previously identified tumor suppressor genes (DLC-1 and DCC) and also for RPIB9 and PCDHGA12. Although the mechanisms that lead to the CGI methylation of these genes are unknown, bisulfite sequencing of the promoter of RPIB9 suggests that expression is inhibited by methylation within SP1 and AP2 transcription factor binding motifs. Finally, specific chromosomal methylation hotspots were found to be associated with ALL. This study sets the stage for acquiring a better biological understanding of ALL and for the identification of epigenetic biomarkers useful for differential diagnosis, therapeutic monitoring, and the detection of leukemic relapse. [Cancer Res 2007;67(6):2617–25]

An intrinsic claudin-low subtype within the TNBC was recently identified based on low/absent expression of luminal differentiation markers, enrichment of epithelial-to-mesenchymal transition (EMT) and stem cell markers. Interleukin-6 (IL6), a potent pleiotropic cytokine, is induced in response to acute and chronic inflammation. IL6 is a key regulator of inflammatory responses and orchestrates these physiological functions by controlling the Suppressor of cytokine signaling 3 (SOCS3) mediated Stat3/NF-κB pathway. SOCS3 is a critical negative regulator of IL6-mediated pathways. We have showed that constitutive activation of inflammatory loop in transformed cells but not in their untransformed counterparts depends on proteolytic degradation of SOCS3. Several studies indicate that activation of the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase (IDO) represents a key pathway suppressing anti-tumor immunity. Moreover a recent study in dendritic cells found that SOCS3 binds IDO via phosphotyrosine-containing peptides and target the IDO/SOCS3 complex for ubiquitination and subsequent proteasomal degradation. IDO is shown to be constitutively expressed by many tumors. We analyzed a panel of breast cancer cell lines and found an inverse correlation between SOCS3 and IDO1 protein levels. Interestingly IDO protein and transcript levels were significantly increased in TNBC compared to luminal cell lines, thus confirming that IL6-JAK-STAT inflammatory loop is responsible for IDO regulation. Enforced expression of stable form of SOCS3, lacking the PEST and SOCS box domain in TNBC cell lines resulted in decreased expression of IDO. Therapeutic targeting of the IDO-IL6-SOCS3 inflammatory loop would revert the immune suppression mediated by IDO in this breast cancer subtype. These studies would provide a strong rationale for development of inflammatory pathway targeted agents for the treatment of TNBC, an aggressive disease that currently lacks molecularly targeted therapeutics. Citation Format: Maria Ouzounova, EunMi Lee, Ena Novakovic, Satish Kumar Noonepalle, Raziye Piranlioglu, Huidong Shi, Max Wicha, Hasan Korkaya. SOCS3 regulates IDO proteasomal degradation and inflammatory signaling in triple negative breast cancer. [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 1984. doi:10.1158/1538-7445.AM2015-1984

Indoleamine 2, 3-dioxygenase 1, encoded by IDO1, is a key immunosuppressive enzyme that catabolizes essential amino acid tryptophan to kynurenine in the tumor microenvironment. Severe depletion of tryptophan by IDO1 affects proliferation of T cells and tryptophan metabolites cause T-cell anergy and induce apoptosis. In this study, we investigated the epigenetic regulation of IDO1 expression by DNA methylation in breast cancer cells. Bisulfite pyrosequencing analysis of IDO1 promoter methylation performed on a panel of 10 breast cancer cell lines revealed that triple negative breast cancer (TNBC) cell lines (i.e. MDA-MB-231, Hs578t, SUM159) are hypomethylated compared to estrogen receptor positive (ER+) cell lines (i.e. MCF7, BT474, T47D). The same analysis was extended to 30 primary breast tumor and normal control samples and the results demonstrated that TNBC tumors had lower IDO1 promoter methylation compared to ER+ tumors. RT-PCR analysis showed that IDO1 mRNA expression is higher in TNBC cell lines than ER+ cell lines. This inverse correlation between IDO1 promoter methylation and mRNA expression can also be observed in TCGA 450K methylation and RNA-seq data sets in which promoter is hypomethylated and mRNA is up-regulated in basal-like molecular subtypes as compared to other breast cancer subtypes. IDO1 promoter is relatively CpG poor with most CpG sites concentrated near interferon γ (IFNg) responsive GAS and ISRE transcription factor binding sites. To investigate the role of CpG methylation in regulating IDO1 induction by IFNg, we cloned either methylated or unmethylated IDO1 promoter DNA into a luciferase reporter plasmid. Methylated promoter reporter exhibited significantly lower luciferase activity at basal or with IFNg stimulation compared to unmethylated reporter plasmid when transfected into MCF-7 or MDA-MB-231 cell lines. Treatment with a demethylating agent, 5-aza-deoxycytidine, synergistically up-regulated IDO1 mRNA expression with IFNg in MCF7 cells which have hypermethylated IDO1 promoter further supporting the influence of CpG methylation on IDO1 expression. IFNg stimulation or co-culture with activated T-cells significantly induced IDO1 protein expression in MDA-MB-231 cells, but not in MCF7 cells. We found no difference in IDO1 mRNA stability and IFNg induced JAK/STAT signaling pathway between MDA-MB-231 and MCF7 cell lines except promoter methylation. Furthermore, RNA-seq analysis of MDA-MB-231 and MCF7 cell lines co-cultured with activated T-cells revealed an active immune signaling mediated through JAK/STAT pathway shared by both cell lines, but also differential induction of IDO1 transcription between these two cell lines. These findings indicate IDO1 promoter methylation status as an important factor that regulates anti-immune responses by tumor cells towards tumor infiltrating lymphocytes and it could be used as a useful biomarker for IDO inhibitor based immunotherapy. Citation Format: Satish Kumar Reddy Noonepalle, Eun Joon Lee, Maria Ouzounova, Jaejik Kim, Jeong-Hyeon Choi, Austin Shull, Lirong Pei, Ravindra Kolhe, Pei-Yin Hsu, Nagireddy Putluri, Tim Hui-Ming Huang, Arun Sreekumar, Hasan Korkaya, David Munn, Huidong Shi. Promoter methylation regulates interferon-γ induced indoleamine 2,3-dioxygenase expression in breast cancer. [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 4060. doi:10.1158/1538-7445.AM2015-4060

Aberrant DNA methylation of promoter CpG islands is associated with transcriptionally repressive heterochromatin in neoplasia. The dynamics of this epigenetic process in mediating the transition from an active to an inactive state of transcription remains to be elucidated, however. Here, we used the methylation-specific oligonucleotide microarray to map the methylation patterns of a CpG island, located within the promoter and the first exon regions of RASSF1A, in normal breast tissue controls, primary tumors, and breast cancer cell lines. Oligonucleotide pairs, spaced along the CpG island region, were designed to discriminate between methylated and unmethylated alleles of selected sites. The methylation-specific oligonucleotide data indicate that the majority of test samples show widespread methylation in the first exon of RASSF1A. In contrast, the promoter area was usually undermethylated in normal controls and in 32% of the primary tumors tested, whereas the rest of the primary tumors and breast cancer cell lines showed various degrees of methylation in the region. Methylation profiling of individual tumors further suggest that DNA methylation progressively spreads from the first exon into the promoter area of this gene. Functional analysis indicates that increased density of RASSF1A promoter methylation is associated with altered chromatin, marked by a depletion of acetylated histones and methylated histone 3-lysine 4 and an enrichment of methylated histone 3-lysine 9 in the studied area. The combination of these epigenetic modifications may engender a stable silencing of the gene in breast cancer cells. Thus, this study underscores the importance of detailed mapping of methylation patterns within a CpG island locus that may provide insights into the progressive nature of aberrant DNA methylation and its relationship with transcriptional silencing during the neoplastic process.

Small interfering RNAs (siRNAs) are newly identified molecules shown to silence genes via targeted mRNA degradation. In this study, we used specific siRNAs as a tool to probe the relationship between two DNA methyltransferase genes, DNMT3b and DNMT1, in the maintenance of DNA methylation patterns in the genome. Levels of DNMT3b or DNMT1 mRNAs and proteins were markedly decreased (up to 80%) on transfecting these siRNAs into the ovarian cancer cell line CP70. The resulting RNA interference showed differential effects on DNA demethylation and gene reactivation in the treated cells. The DNMT1 siRNA treatment led to a partial removal of DNA methylation from three inactive promoter CpG islands, TWIST, RASSF1A, and HIN-1, and restored the expression of these genes. This epigenetic alteration appeared less effective in cells transfected with DNMT3b siRNA. However, the combined treatment of DNMT3b and DNMT1 siRNAs greatly enhanced this demethylation effect, producing 7-15-fold increases in their expression. We also used a microarray approach to examine this RNA interference on 8640 CpG island loci in CP70 cells. The combined siRNA treatment had a greater demethylation effect on 241 methylated loci and selected repetitive sequences than that of the single treatment. Our data thus suggest that whereas DNMT1 plays a key role in methylation maintenance, DNMT3b may act as an accessory to support the function in CP70 cells. This study also shows that siRNA is a powerful tool for interrogating the mechanisms of DNA methylation in normal and pathological genomes.

We developed a novel microarray system to assess gene expression, DNA methylation, and histone acetylation in parallel, and to dissect the complex hierarchy of epigenetic changes in cancer. An integrated microarray panel consisting of 1507 short CpG island tags located at the 5'-end regions (including the first exons) was used to assess effects of epigenetic treatments on a human epithelial ovarian cancer cell line. Treatment with methylation (5-aza-2'-deoxycytidine) or deacetylation (trichostatin A) inhibitors alone resulted in up-regulation of 1.9 or 1.1% of the genes analyzed; however, the combined treatment resulted in synergistic reactivation of more genes (10.4%; P0.001 versus either treatment alone). On the basis of either primary or secondary responses to the treatments, genes were identified as methylation-dependent or -independent. Synergistic reactivation of the methylation-dependent genes by 5-aza-2'-deoxycytidine plus trichostatin A revealed a functional interaction between methylated promoters and deacetylated histones. Increased expression of some methylation-independent genes was associated with enhanced histone acetylation, but up-regulation of most of the genes identified using this technology was because of events downstream of the epigenetic cascade. We demonstrate proof of principle for using the triple microarray system in analyzing the dynamic relationship between transcription factors and promoter targets in cancer genomes.

We present a novel concept by using expressed CpG island sequence tags (ECISTs)for dual analysis of DNA methylation and gene expression in cancer cells. ECISTs are present in the genome and are DNA fragments expected to be located in the promoter and first exon region of genes. Their GC-rich segments can be used for screening hypermethylated CpG sites in cancer cells, and their exon-containing portions can be used for measuring levels of the corresponding transcripts. A total of 1162 loci met the criteria of ECISTs from an initial screening of 7776 CpG island tags. This ECIST panel was used to analyze the breast cancer cell line MDA-MB-231, which was treated with a demethylating agent. Microarray profile analysis identified 30 methylation-silenced genes, the expression of which could be directly reactivated by demethylation. An additional group of 96 up-regulated genes was also identified but appeared to be downstream from this epigenetic cascade. Thus, this study shows that the ECIST microarray can be used to differentiate the primary and secondary causes of demethylation and provides an effective tool to elucidate the mechanisms of aberrant DNA methylation in cancer.

Metastasis is the leading cause of caner-related death for solid tumors including gastric carcinoma (GC). An epigenetic biomarker panel for prediction GC metastasis will have clinical impact on the care of GC patients. In order to characterize the methylation differences between GCs with and without metastasis, genome-wide DNA methylation profiles were compared between 4 metastatic and 4 non-metastatic GCs and their surgical margins (SM). The GC genome showed significantly higher proportions of hypomethylation in the promoter and exon-1 regions, as well as increased hypermethylation of intragenic fragments when compared to SMs. From the list of differentially methylated CpG islands (CGIs), 63 candidate genes and 10 known tumor-related genes were selected for further analysis based on their known functions. Significant differential methylation was validated in the CGIs of 15 genes (Ps Citation Format: Zhaojun Liu, Jun Zhang, Yanhong Gao, Lirong Pei, Naoko Hattori, Budong Zhu, Jiafu Ji, Yasuhito Yuasa, Wooho Kim, Toshikazu Ushijima, Huidong Shi, Dajun Deng. Large-scale characterization of DNA methylation changes in human gastric carcinomas with and without metastasis. [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 4012. doi:10.1158/1538-7445.AM2014-4012

Glioblastoma (GBM) is the most common and most aggressive brain tumor in adults. Its frequent recurrence after resection and dismal prognosis are thought to be due to a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy. In this study, we performed an in depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary GBM tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified two groups hyper- and hypomethylated of genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also be found in primary GBM-derived xenograft tumors suggesting that they were not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down regulated in GBMs such as SPINT2, NEFM, and PENK. The comparison between the NSC line and the normal brain tissue sample leads to the identification of a group of NSC-specific differentially methylated regions (nsDMRs). Cluster analysis using nsDMRs revealed the presence of stem cell-specific DNA methylation signatures in both primary GBM and GSC lines. Higher expression of genes associated with stem cell-specific DNA methylation signatures such as DNMT3A, STAT5A, CSTB, PMEPA1 and G6PD in GBM patients was found to be associated with poor patient survival. The results from this study demonstrate the utility of using cancer stem cell models for advancing understanding of the pathobiology of gliomas. Citation Format: Eun Joon Lee, Prakash Rath, Jimei Liu, Dungsheng Ryu, Alan Free, Lirong Pei, Douglas C Anthony, Suash Sharma, Mark D Kirk, John J. Laterra, Duck Hwan Ryu, Jeong-Hyeon Choi, Huidong Shi, Douglas C. Miller, N. Scott Litofsky, Qi Feng. Identification of global DNA methylation signatures in glioblastoma-derived cancer stem cells. [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 1379. doi:10.1158/1538-7445.AM2014-1379

The structure of chromatin is tightly regulated; its disregulation is causal for developmental abnormalities and for cancer. The nucleosome remodeling and deacetylase (NuRD) complex regulates chromatin structure and gene expression via its nucleosome remodeling and histone deacetylase activities. MBD3, a component of NuRD, has pleiotropic roles in embryonic stem (ES) cells and tumor biology. However, the results of recent studies are conflicting, resulting in an unclear model for NuRD function. Here, we investigated the localization of MBD3 in two human breast cancer cell lines (MCF-7 and MDA-MB-231) with two independent genomic technologies (DamID and ChIP-seq). DamID and ChIP-seq data were in an excellent agreement and led consistent conclusions in both cell lines. MBD3 localized not only at promoters and enhancers of active genes but also accumulated at gene bodies; this previously undescribed localization suggests an active role of MBD3 in transcription. MBD3 preferred hypomethylated, CpG rich promoters marked by H3K4me3. We also noted a subset of MBD3 that localized at silent promoters marked by H3K9me3 or H3K27me3, consistent with biochemical studies. Another subset of MBD3 co-localized with H3K27ac and was in physiological proximity with promoters, suggesting a role of MBD3 in the regulation of high-order structure of the genome. Functional analysis of chromatin demonstrated that MBD3 regulates nucleosome occupancy near promoters and in gene bodies. These data suggest multiple roles of MBD3/NuRD in the regulation of chromatin structure and gene expression for both active and silent genes. The findings serve as an important initial step in defining regulatory mechanism of MBD3/NuRD in ES cells and in tumor biology. Citation Format: Takashi Shimbo, Ying Du, Sara A. Grimm, Archana Dhasarathy, Deepak Mav, Ruchir R. Shah, Huidong Shi, Paul A. Wade. Mbd3 localizes at promoters, gene bodies and enhancers of active genes. [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 405. doi:10.1158/1538-7445.AM2014-405

Chronic lymphocytic leukemia (CLL) is a cancer in which B-lymphocytes proliferate in an unchecked manner and escape normal apoptotic death. It is one of the most common leukemias in the Western world, and currently, there is no cure for CLL due to the heterogeneous ways in which it can proliferate. Due to the dynamics of how CLL can develop, it is very critical to investigate the oncogenic mechanisms that allow particular clinical CLL subtypes to expand, as well as discovering potential underlying mechanisms that are common among all CLL patients. With this comprehensive goal in mind, we investigated the gene expression landscape in CLL by performing whole transcriptome analysis via RNA sequencing in 47 CLL patients and 5 normal CD19+ B-cell donors to determine differential expression and isoform patterns in CLL. Between CLL and normal CD19+ B-cells, there were 725 differentially expressed genes (FDR p Citation Format: Austin Y. Shull, Junfeng Luo, Jeong-Hyeon Choi, Lirong Pei, Farrukh T. Awan, Eun-Joon Lee, Jimei Liu, Phillip J. Buckhaults, Xiao-Jie Yan, Nicholas Chiorazzi, Huidong Shi. Identifying differential gene expression and splicing events in chronic lymphocytic leukemia patients through whole transcriptome profiling. [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 5182. doi:10.1158/1538-7445.AM2014-5182

The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. Recent reports describing localization of NuRD are not in complete agreement and provide new insights that question previous models on NuRD action. Here, we provide location analysis of endogenous MBD3, a component of NuRD complex, in two human breast cancer cell lines (MCF7 and MDA-MB-231) using two independent genomic techniques: DNA adenine methyltransferase identification (DamID) and ChIP-Seq. Contrary to existing models, MBD3 preferentially associated with CpG rich promoters marked by H3K4me3 and lacking 5-methyl C or 5-hydroxymethyl C. MBD3 also showed cell-type specific localization across gene bodies, peaking around the transcription start site (TSS). A subset of sites bound by MBD3 was enriched in H3K27Ac and was in physical proximity to promoters in three dimensional space, suggesting function as enhancers. MBD3 enrichment was also noted at promoters modified by H3K27me3. These data suggest that MBD3, and by extension the Mi-2/NuRD complex, may have multiple roles in fine tuning expression for both active and silent genes. These data represent an important step in defining regulatory mechanisms by which Mi-2/NuRD complex controls chromatin structure and modification status. Citation Format: Takashi Shimbo, Ying Du, Sara Grimm, Archana Dhasarathy, Deepak Mav, Ruchir Shah, Huidong Shi, Paul A. Wade. MBD3 accumulates at promoters and enhancers of active genes. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B63.

Chronic lymphocytic leukemia (CLL) is the most common leukemia of adults in the western world. The clinical course of CLL is highly variable. Some patients are able to live years with no need for treatment; whereas others progress rapidly requiring therapy within a short time after diagnosis. The identification and validation of prognostic molecular markers (including surface markers, cytogenetic abnormalities, and IGHV mutational status) have resulted in refinements in the approach to the management of these patients. Further discovery of biologically relevant factors that influence the heterogeneity and progression of CLL will not only promote our understanding of the disease process, but also allow us to identify rational therapeutic approaches. In this study, we conducted genome-wide DNA methylation analyses in purified CD19+ B-cells from 11 CLL patients with a range of CD38 expression using reduced representation bisulfite sequencing (RRBS). Using one lane of the Illumina sequencing data, we were able to consistently determine the methylation status of approximately 1.8 million CpGs; 45% of these CpGs are located in more than 23,000 CpG islands (CGIs), accounting for more than 80% of all annotated CGIs across the genome. We have identified a large number of CGIs that were differentially methylated between CLL cells and normal CD19+ B-cells. The promoter hypermethylation patterns of many genes previously reported in CLL, such as FOXD3, GRM7, DLEU7 etc, were determined in the 11 CLL samples. In addition, we have observed aberrant DNA methylation changes in all 4 HOX gene clusters in CLL. For instance, HOXD8, HOXD9, and HOXD11 were hypermethylated in 11 out of 11 CLL samples. In addition, we also identified a significant number of DNA methylation alterations in the WNT signaling pathway genes. One-way ANOVA analysis of DNA methylation profiles of 764,984 CpGs shared by all CLL samples identified 570 CpGs that were differentially methylated (p20% CLL cells expressing CD38 as CD38high group). The cluster analysis of the 570 CpGs reveals that it is possible to separate these CLL samples into two distinct groups based on the DNA methylation profiles. The study further confirms our early findings that CLL is affected by CpG island methylation in some genes that segregate with CD38 expression levels, while most others show similar methylation patterns across all levels. The aberrant promoter hypermethylation in certain functional gene groups and pathway-associated genes that are known to be deregulated in CLL provides additional insights into the CLL methylome and epigenetic contribution to cellular dysfunction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4791. doi:10.1158/1538-7445.AM2011-4791

Recent studies using next-generation sequencing have generated genome-wide, single-base resolution DNA methylation maps. However, it is still very costly to conduct whole genome shotgun bisulfite sequencing. Currently, few methods enable targeted bisulfite sequencing of CpG islands (CGIs) or other specific regions of interest in a highly flexible and efficient manner. Here we present a novel approach that combines solution-phase hybrid selection and massively parallel bisulfite sequencing to profile DNA methylation in targeted CGI and promoter regions. We designed 51,466 single strand DNA oligonucleotides (160-mer) which target 23,441 CGIs and the transcription start sites of 19,369 known genes in the human genome. The synthetic long DNA oligonucleotides were converted into biotinylated RNA probes for solution-phase hybridization capture of target DNA. The captured genomic DNA was treated with sodium bisulfite, amplified by PCR and sequenced using Illumina GA IIx sequencer. Using this approach, we conducted bisulfite sequencing on captured DNA from three breast cancer cell lines, MCF10A, MCF7, and MDA-MB-231. 20-30 million single-end 75bp sequencing reads were obtained for each cell line. The raw sequencing reads were mapped to in silico bisulfite-converted human genome. The methylation levels for CpG sites covered with at least 10 sequencing reads were extracted, providing accurate quantification on 900,000-1,000,000 CpGs. 77-84% of CpGs analyzed in these samples fell on or near capture probe sequences; 69-75% lay properly on CGIs. One lane of Illumina sequencing data was sufficient to determine the methylation status of over 22,000 CGIs (75% of all annotated CGIs) in the human genome. More than 85% of capture probes successfully yielded quantitative DNA methylation data of targeted regions. We chose 45 candidate loci (760 CpGs) for confirmation with PCR-based bisulfite sequencing and demonstrated excellent correlation between two data sets. This novel method was further validated by sequencing three DNA methyltransferase (DNMT) knockout cell lines and the parental HCT116 colon cancer cell line. The targeted bisulfite sequencing data shows massive demethylation events in DNMT1 knockout (1KO) and double knockout (DKO) cell lines, but not in DNMT3B knockout (3BKO) and HCT116 cell lines. While the results indicate the critical role of DNMT1 in maintaining global DNA methylation, we have also identified DNMT3B specific demethylation patterns in a small group of genomic loci. In this study, we demonstrated the targeted bisulfite sequencing approach to be a powerful method to uncover novel aberrant methylation in the cancer genome. Since all targets were captured and sequenced as a pool through a series of single-tube reactions, this method can be easily scaled up to deal with a large number of samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3003. doi:10.1158/1538-7445.AM2011-3003

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that regulate gene expression by translational repression or mRNA degradation. Although the functions of specific miRNAs in breast cancer development and progression have been extensively investigated, few studies have focused on the systematic identification of miRNAs and their target genes related to breast cancer aggressiveness. In this study, we sought to discover the signatures of miRNA-mRNA target pairs in aggressive breast cancer cell lines through a combination of integrated miRNA and mRNA expression profiling, bioinformatics prediction and functional assays. MiRNA and mRNA expression profiles of 12 breast cancer cell lines including 8 less aggressive lines (MCF10A, MCF12A, BT474, MCF7, MDA-MB-468, SK-BR3, T-47D and ZR-75-1) and 4 aggressive lines (BT549, HS578T, MDA-MB-231 and SUM159) were generated using Affymertrix miRNA Array and U133 Plus 2.0 Array, respectively. The miRNA expression array analysis has shown that 44 out of 847 miRNAs were differentially expressed (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4937. doi:10.1158/1538-7445.AM2011-4937