Abstract Purpose: In preclinical studies, the lysine-specific histone demethylase 1A (LSD1) inhibitor tranylcypromine (TCP) combined with all-trans retinoic acid (ATRA) induces differentiation and impairs survival of myeloid blasts in non-acute promyelocytic leukemia acute myeloid leukemia (AML). We conducted a phase I clinical trial (NCT02273102) to evaluate the safety and activity of ATRA plus TCP in patients with relapsed/refractory AML and myelodysplasia (MDS). Patients and Methods: Seventeen patients were treated with ATRA and TCP (three dose levels: 10 mg twice daily, 20 mg twice daily, and 30 mg twice daily). Results: ATRA-TCP had an acceptable safety profile. The MTD of TCP was 20 mg twice daily. Best responses included one morphologic leukemia-free state, one marrow complete remission with hematologic improvement, two stable disease with hematologic improvement, and two stable disease. By intention to treat, the overall response rate was 23.5% and clinical benefit rate was 35.3%. Gene expression profiling of patient blasts showed that responding patients had a more quiescent CD34+ cell phenotype at baseline, including decreased MYC and RARA expression, compared with nonresponders that exhibited a more proliferative CD34+ phenotype, with gene expression enrichment for cell growth signaling. Upon ATRA-TCP treatment, we observed significant induction of retinoic acid–target genes in responders but not nonresponders. We corroborated this in AML cell lines, showing that ATRA-TCP synergistically increased differentiation capacity and cell death by regulating the expression of key gene sets that segregate patients by their clinical response. Conclusions: These data indicate that LSD1 inhibition sensitizes AML cells to ATRA and may restore ATRA responsiveness in subsets of patients with MDS and AML.

Abstract Introduction: Tranylcypromine (TCP) is an irreversible monoamine oxidase inhibitor, a potent antidepressant that has been in use since the 1960s. Additionally, TCP has been demonstrated to inhibit lysine-specific histone demethylase 1A (LSD1), which is highly expressed in AML (Lee 2006; Berglund 2008). Preclinical studies combining TCP and ATRA induced differentiation and impaired clonogenic survival in non-APL AML cell lines and primary patient samples. These findings were supported by mouse xenograft models (Schenk 2011). Based on this preclinical work, we pursued an investigator-initiated Phase 1 study of this combination at the University of Miami Sylvester Comprehensive Cancer Center (NCT02273102). Methods: A Phase 1 study was initiated to evaluate the safety, PK/PD, and preliminary clinical activity of TCP in combination with ATRA in patients (pts) with relapsed/refractory AML and high-grade MDS. The study followed a traditional 3+3 dose escalation design. Safety for all pts and efficacy for all evaluable pts to date are reported. All adverse events were recorded per NCI CTCAE v4.03. All pts received continuous daily dosing of both ATRA (45 mg/m2 in divided doses) and TCP (3 escalating dose levels: 10mg BID, 20mg BID and 30mg BID), with a 3-day lead-in of TCP only during cycle 1. Cycles were 21 days and pts were allowed to remain on study until progression or unacceptable toxicity. Results: At the time of data cutoff, 15 pts had received therapy with combination TCP/ATRA (8 AML and 7 MDS). Median age was 74 years, 40% were female and 67% white/27% Hispanic/7% black. Overall, the combination was well tolerated, with the majority of treatment emergent adverse effects (TEAEs) Grade 1 and 2. The most common TEAEs (all grades, ≥20%) included dry mouth (33%), dry skin (27%), febrile neutropenia (27%), dizziness (27%), fatigue (27%), headache (27%), rash (27%), increase in creatinine (27%); and vomiting, nausea, diarrhea, infection, urinary frequency and thrombocytopenia all with a frequency of 20%. The most common Grade 3/4 TEAEs included febrile neutropenia (27%), thrombocytopenia (20%), sepsis (13%), lung infection (13%) and anemia (13%). There was 1 DLT of dizziness at the TCP 20mg BID dose level (out of 8 pts) and 2 DLTs of generalized weakness and nausea/vomiting, respectively, at 30mg BID (out of 3 pts). All DLTs were grade 2, but persistent and poorly tolerated. Therefore, TCP 20mg BID was determined to be the MTD and selected as the RP2D. Best evaluable responses per modified IWG/ELN criteria included 5 pts with prolonged stable disease (all 3 months or more) (2 AML, 1 CMML, 2 MDS), 1 marrow CR (MDS) and 1 MLFS (AML). Three of the 4 MDS/CMML responders had hematologic improvement (HI) (2 HI-P and 1 HI-P and HI-E). One AML pt also recovered neutrophils (0.62 to 14.75) with a decrease in blasts but did not meet response criteria. The 2 pts with best response of marrow CR and MLFS continued on study for 7 and 10 months, respectively. Importantly, these 2 pts and a third pt who had prolonged SD (5 months) plus HI-P/HI-E were all taken off study for cumulative skin toxicity (not progression), and the marrow CR and MLFS pts are both still alive. Conclusions: TCP/ATRA combination therapy has demonstrated an acceptable safety profile in pts with R/R AML and MDS, and additionally has demonstrated clinical activity. TCP 20mg BID is the RP2D, and a phase 1 dose expansion at this dose level is ongoing. In responders, skin toxicity may be treatment duration-limiting due to continuous exposure to ATRA, and an intermittent ATRA schedule after cycle 4 may be pursued for the phase 2 study. Additional data will be presented at the meeting, including myeloid mutational analysis, RNA-seq and ATAC-seq, in order to delineate pre- and post-treatment molecular profiles and chromatin accessibility in these pts. Preliminary data (not shown) suggest that a baseline gene expression pattern may predict sensitivity or resistance to TCP/ATRA. Disclosures Watts: Takeda: Research Funding; Jazz Pharma: Consultancy, Speakers Bureau. Swords:AbbVie: Employment.

Abstract Cancer cell invasion is an obligatory step for metastatic dissemination that contributes to rapid relapse and a poor survival in TNBC patients. Development of novel therapeutic strategies to block tumor invasion is an unmet need for TNBC treatment and for other tumor types. We reported that decoys with the SID sequence designed to bind and inhibit the function of PAH-2 domain of Sin3A protein markedly prolong survival in the adjuvant setting due to inhibition of metastatic dissemination to the lungs and bone marrow in TNBC mouse models. Here, we show that TNBC cell lines treated with SID decoys (peptides) display a strong in vitro inhibition of migration and invasion. This is accompanied by actin cytoskeleton reorganization with increased cortical actin, and inhibition of proteolytic enzymes (MMP9; MT-MMP1 and uPA) involved in extracellular matrix degradation. DNA microarray and Ingenuity pathway analysis (IPA) showed that the SID decoys inhibit Wnt and TGFβ signaling that is associated with epithelial to mesenchymal transition (EMT). Treatment with SID decoy peptide downregulated WNT/β-catenin-driven transactivation as measured by decreased promoter H3K4me3 and decreased expression of Wnt target genes like LEF1 and TCF7L2. We also show that SID decoys induce translocation of nuclear β-catenin to the cytoplasm in TNBC at 24 hours. Wnt/β-catenin is critical for EMT, cancer stem cell self-renewal, and early invasion in TNBC. TGIF1, a transcription factor that modulates TGFβ and Wnt signaling pathways and known to to interact with the PAH2 domain of Sin3A, can be dissociated from Sin3A complex by SID decoy treatment as measured by co-immunoprecipitation (Co-IP) and proximity linked assay. DNA microarray of SID peptide treated TNBC cells shows inhibition of TGFβ signaling evidenced by downregulation of MMP9, MT1-MMP and PLAU, known target genes of this pathway. This is in line with inhibition of the EMT program predicted by the IPA analysis in SID peptide treated TNBC. Taken together, the results indicate that SID decoys have potential value as therapeutic agents to revert the EMT program in TNBC that should translate into the inhibition of metastasis dissemination and eradication of residual disease in TNBC. To test this in clinic future investigations will involve the use of our previously identified small molecule mimetic of SID peptide, selamectin that is also a FDA approved drug. Use of a recently constructed cyclic stapled peptide that inhibits PAH-2 binding and invasion at <10nM is also anticipated. Citation Format: Yeon-Jin Kwon, Boris A. Leibovitch, Nidhi Bansal, Lutecia Pereira, Edgardo V. Ariztia, Kevin Petrie, Arthur Zelent, Ming-Ming Zhou, Eduardo F. Farias, Samuel Waxman. Inhibition of triple negative breast cancer cell invasion by the targeted interference of Sin3A function affecting Wnt and TGFβ signaling. [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 4115.

Abstract Resistance to chemotherapy and subsequent relapse is the most challenging issue in the treatment of patients with Acute Myeloid Leukemia (AML). However, the underlying mechanisms still remain incompletely understood. Here we report that loss of the histone methyltransferase EZH2 and subsequent reduction of H3K27 trimethylation contribute to chemoresistance in AML. In Myelodysplastic Syndrome (MDS) and Myeloproliferative Neoplasms (MPN) EZH2 is often inactivated due to mutations which is associated with poor prognosis. By use of quantitative PCR and immunohistochemistry we show that a decrease of EZH2 mRNA and protein also correlated with a poor prognosis of AML patients indicating a tumor suppressor role of EZH2 in AML. EZH2 is located on chromosome 7q36.1 and it is not yet fully clear whether EZH2 expression is affected in MDS and AML patients with del(7)/del(7q) who are largely refractory to chemotherapy and have a poor prognosis. We found EZH2 levels to be reduced in del(7)/del(7q) AML patients as determined by Western Blot. Notably, the reduction of EZH2 protein levels via treatment with H3K27 methyltransferase inhibitors or lentiviral knockdown was sufficient to induce chemoresistance of Normal Karyotype (NK)- AML blasts and cell lines in vitro and in a xenograft mouse model. Furthermore, we observed that EZH2 loss occurred during the acquisition of drug resistance in a Tyrosine Kinase Inhibitor- and Cytarabine (AraC)- resistant AML cell line. Pharmacological inhibition of CDK1 and treatment with the proteasome inhibitor Bortezomib, respectively, increased EZH2 protein and restored drug sensitivity. Functionally, the loss of EZH2 directly induced upregulation of HOX genes, suggesting a stem-cell-like signature to be associated with the resistance phenotype, which could be reverted by Bortezomib treatment. To evaluate the potential of Bortezomib to affect EZH2 levels in patient blasts we treated primary NK-AML blasts collected at diagnosis ex vivo with Bortezomib. In almost all samples Bortezomib treatment induced cytotoxic effects. In 5 out of 10 patients the EZH2 protein level could be increased by Bortezomib treatment. We furthermore examined the sensitivity of patient samples to AraC, Bortezomib or combined treatment. Notably, for those patients with increased EZH2 levels after Bortezomib exposure we found a significantly decreased cell survival for the combined treatment compared to single-agent treatment. Our data strongly suggest that restoration of EZH2 protein levels e.g. via proteasome inhibitors and thereby restoration of EZH2 function might be a novel promising approach to increase therapy response in AML. Citation Format: Stefanie Göllner, Shuchi Agrawal-Singh, Tino Schenk, Hans-Ulrich Klein, Christian Rohde, Tim Sauer, Mads Lerdrup, Sigal Tavor, Friedrich Stölzel, Gerhard Ehninger, Gabriele Köhler, Martin Dugas, Arthur Zelent, Christian Thiede, Wolfgang E. Berdel, Klaus Hansen, Carsten Müller-Tidow. Loss of the histone methyltransferase EZH2 induces chemoresistance in acute myeloid leukemia (AML). [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 4430. doi:10.1158/1538-7445.AM2015-4430

Acute Promyelocytic Leukemia (APL) accounts for 5% of all cases of acute myeloid leukemia (AML). This disease is highly curable with all-trans-retinoic acid (ATRA) based therapy. In non-APL AML, ATRA has limited activity, and little is known about mechanisms of ATRA resistance. The apparent selective efficacy of ATRA in PML/RARα-associated APL poses an important question as to whether the presence of this fusion protein renders APL uniquely susceptible. Two compelling arguments can be made to counter this view. First, experiments in vitro show that ATRA effectively differentiates HL-60 cell lines, which lack the PML/RARα fusion protein. Second, clinical studies with ATRA in previously untreated older AML patients (excluding APL) have reported clinical activity. These observations confirm the therapeutic potential of ATRA beyond APL. In this context, our group has previously identified the lysine demethylase LSD-1, as a therapeutic target to re-sensitize leukemic blasts to ATRA. A clinical investigation of ATRA combined with LSD-1 inhibition is currently underway (NCT02273102). It is likely that other defects leading to ATRA resistance will be similarly amenable to pharmacologic manipulation. Defects in the proto-oncogene c-Myc have been widely implicated in the initiation and maintenance of AML. Over-expression of c-Myc in leukemic blasts enhances clonogenic survival and blocks ATRA induced differentiation. We hypothesized that down-regulation of c-Myc might increase the anti-leukemic effects of ATRA in AML. To date, c-Myc has been an evasive target for direct pharmacologic inhibition however, inhibitors of the PI3K/AKT/mTOR pathway have been shown to indirectly lower levels of c-Myc in leukemic blasts.

The histone methyltransferase Enhancer of Zeste Homologue 2 (EZH2), a component of the polycomb group complex, is critical for normal hematopoietic stem cell development. EZH2 mediates transcriptional repression through histone tri-methylation (H3K27me3). The activity of EZH2 influences cell fate regulation, namely the balance between self-renewal and differentiation. The contribution of aberrant EZH2 expression to tumorigenesis is becoming increasingly recognized. Its role in hematological malignancies however, is complex. Both gain-of-function and loss-of-function mutations have been respectively reported in lymphoma and leukemia, suggesting that EZH2 may serve a dual purpose as an oncogene and tumor-suppressor gene. Impaired self-renewal via EZH2 inhibition has been observed and offers a potentially attractive therapeutic approach in acute myeloid leukemia. Indeed, overexpression of EZH2 has been reported in patients with AML, particularly in those with complex karyotypes. In the present study, we show that deletion of EZH2 compromises the growth potential of AML cells by promoting their differentiation. To understand the role of EZH2 in vitro, we first examined the cell growth and colony-forming ability of EZH2 knockdown vs WT HL-60 cells. We found that proliferation of HL-60 cells was severely compromised following deletion of EZH2. Additionally, EZH2 deletion resulted in retarded cell-cycle entry and resulted in increased apoptotic cell death Similarly, the number of total colonies generated by EZH2 deleted cells in the secondary and tertiary re-plating assays was considerably less than that of controls. EZH2 deleted cells tended to form dispersed colonies that were mainly composed of differentiated myeloid cells, whereas control cells mostly formed compact colonies composed of myeloblasts. The proportion of dispersed colonies in the EZH2deleted cell culture increased with serial replatings. Deletion of EZH2 affects the growth and replating capacity of AML cell in vitro. When EZH2 deleted HL-60 cells were treated with the retinoid all-trans-retinoic acid (ATRA), we observed a marked induction of differentiation (as measured by the myeloid maturation marker CD11b) compared to the effects of ATRA on differentiation in wild type (WT) cells. Similarly, impaired clonogenic survival was more pronounced following ATRA treatment in EZH2 deleted vs WT HL-60 cells (see figure). We then profiled a number of small molecule inhibitors of EZH2 alone (EPZ005687, EPZ-6438, GSK126, El1, DZNeP, UNC1999 and GSK343) and in combination with ATRA, confirming these phenotypic changes. To elucidate the mechanism for how EZH2 regulates the balance of self-renewal vs differentiation in AML, we examined the genome-wide distribution of H3K27me3 by ChIP-seq analysis. First, western blot analysis revealed a marked decrease in the levels of H3K27me3 in EZH2 deleted AML cells. Next, we examined the presence of H3K27me3 marks in leukemia cells purified by ChIP-seq analysis. We focused on the region from 5.0 kb upstream to 3.0 kb downstream of transcription start sites (TSSs) of reference sequence (RefSeq) genes (http://www.ncbi.nlm.nih.gov/RefSeq/) because H3K27me3 marks are usually enriched near TSSs or across the body of genes. As expected, the deletion of EZH2 caused a drastic reduction in these H3K27me3 marks. Targeting EZH2 presents and interesting dichotomy as a novel drug target since inhibition of this protein could potentially be beneficial or detrimental depending on the context of the disease. In the case of AML, EZH2 mutations likely impede differentiation and block retinoic acid led differentiation programs. Updated studies outlining the interaction between the retinoic acid signaling pathway and EZH2 will be presented. These studies justify clinical investigation of EZH2 inhibitors combined with ATRA for patients with AML. Disclosures No relevant conflicts of interest to declare.

Introduction - With rapidly advancing sequencing technology, the extent of genetic diversity in AML has never been more apparent. A Òone size fits allÓ approach can no longer be justified. Sequencing studies have also uncovered several actionable targets, yet no targeted therapies are FDA approved for use in the US. AML therefore, has significant potential for personalized therapeutics. Several challenges exist, however. The masses of data generated by high-throughput technologies are challenging to manage, visualize, and convert to knowledge required to improve outcomes. A cross-disciplinary systems biology effort is required, to visualize inter-connected events within leukemic blasts that ultimately contribute to the disease phenotype and inform on rational selection of therapeutic approaches. In the current study, we outline a complimentary functional and genomic screening approach to identify clinical drug candidates for re-purposing in patients with relapsed refractory AML.

Abstract Introduction: Dearth of clinically validated drug targets is a major challenge in Triple Negative Breast Cancer (TNBC) that limits the treatment options to chemotherapy with intense cytotoxic consequences. We previously identified PAH2 domain of Sin3 protein as potential therapeutic target in TNBC and shown that protein-protein interactions of Sin3 via its PAH-2 domain can be disrupted by decoys (Mad1-SID peptide/small molecule inhibitors) designed based on the Sin3 interaction domain (SID) of transcription factor Mad1. Chromatin regulator Pf1 shown to be overexpressed in breast cancer and interacting with PAH2 domain of Sin3 can also be dissociated from Sin3 complex by Mad1-SID. In this study we identify Pf1 to be a significant contributor to the oncogenic phenotype associated with TNBC. Methods: Pf1 knockdown lines were generated by stably transfecting MDA-MB-231 cells with Pf1-shRNA or scr-shRNA. Cells were cultured in 2D and assayed for cancer stem cell (CSC) markers and ALDH activity by flow cytometry. For colony morphogenesis cells were cultured in 3D Matrigel. qRT-PCR were performed to assay expression of Nanog, Sox2 and Oct4. Fluorescence anisotropy and co-immunoprecipitation assays were used to test Sin3-Mad1 and Sin3-Pf1 interaction in the presence or absence of Mad1-SID and/or Pf1-SID. Results: MDA-MB-231 cells transfected with Pf1-shRNA had over two fold reduced ability to form colonies in 3D Matrigel cultures, with ∼95% of colonies non-invasive in contrast to the invasive star-like colonies formed by cells transfected with Scr-shRNA. Additionally reduction in Pf1 level was accompanied by a ∼1.5-fold reduction (p<0.05) in the tumorsphere forming ability of MDA-MB-231 cells. Pf1 depletion also significantly reduced RNA and protein of Nanog, Oct4 and Sox2. Confocal imaging revealed reduced levels and nuclear accumulation of Nanog, Sox2 and Oct4 in cells transfected with Pf1-shRNA compared to Scr-shRNA. Pf1 knockdown resulted in a 2.5-fold decrease in ALDH1 positive cells (6.55% in Scr-shRNA vs 2.59% in Pf1-shRNA). The CD44low/CD24low/neg population was enriched 3 fold over cells transfected with Scr-shRNA. From a therapeutic perspective we have designed a linear peptide corresponding to the Sin3 interaction domain of Pf1 (Pf1-SID). By fluorescence anisotropy we show that in comparison to Mad1-SID (IC50 = 1.4±0.3 μM), several hundred times higher concentration of Pf1-peptide (IC50 = 826±162 μM) is required to dissociate Sin3-Mad1 interaction. Co-immunoprecipitation assays show that Pf1-SID can specifically dissociate Pf1 from Sin3 without affecting the binding of Mad1 with PAH2 domain of Sin3. Pf1-SID treated MDA-MB-231 cells also have reduced invasive capacity and CSC traits. Conclusion: PAH2 domain of Sin3 and its interaction with Pf1 is potential drug target and Pf-1SID-mediated disruption of Sin3-Pf1 complex as translation relevance for first site-specific epigenetic therapy for TNBC. Citation Format: Nidhi Bansal, Joanna Wexler, Yeon-jin Kwon, Elena C. Gil, Boris Leibovitch, Rajal Sharma, Arthur Zelent, Ming-Ming Zhou, Eduardo Farias, Samuel Waxman. Targeting Sin3-Pf1 complex: Novel site-specific epigenetic therapy for 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 1989. doi:10.1158/1538-7445.AM2015-1989

Triple negative breast cancer (TNBC) is characterized by a poorly differentiated phenotype and limited treatment options. Aberrant epigenetics in this subtype represent a potential therapeutic opportunity, but a better understanding of the mechanisms contributing to the TNBC pathogenesis is required. The SIN3 molecular scaffold performs a critical role in multiple cellular processes, including epigenetic regulation, and has been identified as a potential therapeutic target. Using a competitive peptide corresponding to the SIN3 interaction domain of MAD (Tat-SID), we investigated the functional consequences of selectively blocking the paired amphipathic α-helix (PAH2) domain of SIN3. Here, we report the identification of the SID-containing adaptor PF1 as a factor required for maintenance of the TNBC stem cell phenotype and epithelial-to-mesenchymal transition (EMT). Tat-SID peptide blocked the interaction between SIN3A and PF1, leading to epigenetic modulation and transcriptional downregulation of TNBC stem cell and EMT markers. Importantly, Tat-SID treatment also led to a reduction in primary tumor growth and disseminated metastatic disease in vivo. In support of these findings, knockdown of PF1 expression phenocopied treatment with Tat-SID both in vitro and in vivo. These results demonstrate a critical role for a complex containing SIN3A and PF1 in TNBC and provide a rational for its therapeutic targeting.

Aberrant epigenetics leading to changes in chromatin structure and patterns of gene expression is an important factor in cancer pathogenesis. Histone Deacetylase 9 (HDAC9) is a class IIa chromatin-modifying enzyme that, within the haematopoietic system, is preferentially expressed in the B-cell lineage. Mice that constitutively express human HDAC9 from early stages of B-cell development, under the control of the immunoglobulin heavy chain (IgH) enhancer, develop lymphoproliferative disorders, including germinal center (GC) and post-GC lymphomas, demonstrating an oncogenic role for HDAC9 in B-cells and highlighting its importance as a therapeutic target. In order to examine the relationship between disease observed in the mouse model and human primary lymphoma, we have examined, using immunohistochemistry (IHC) the expression of full length HDAC9 isoform in a panel of various B-cell malignancies. The study group included 59 non-Hodgkin lymphomas (NHL), and 3 classical HL. Non-HL consisted of 34 diffuse large B cell lymphoma (DLBCL), 9 follicular lymphoma (FL), 5 marginal zone lymphoma (MZL), 6 mantle cell lymphoma (MCL), and 2 small lymphocytic lymphomas (SLL). HDAC9 expression was assessed by IHC using tissue microarray and/or routine tissue sections. Protein expression was scored as negative (0), low (1), or high (2) depending on the staining signal intensity. Expression of HDAC9 in the nuclei of the tumor cells was compared with that seen in adenocarcinoma cells; if equal or higher, then expression of HDAC9 was considered high and if lower, then expression of HDAC9 was considered low. Five reactive lymph nodes were studied to assess the baseline expression of HDAC9. Rectal adenocarcinomas were used as positive controls. In reactive lymph nodes, HDAC9 was weakly expressed in a subset of germinal center cells, a subset of lymphoid cells in the paracortex as well as in endothelial cells. HDAC9 expression was detected in all subsets of B-cell lymphomas analyzed and in most cases with a level of expression higer than those seen in reactive lymph nodes. DLBCL and MCL tumors have the highest frequency of high HDAC9 expression among the B-cell lymphomas analyzed, 77 and 83% (Fisher’s exact test P=1,0), respectively. No differences in HDAC9 expression were detected in DLBCL of GC and non-GC type. In contrast, most (69%) of the low-grade B cell lymphomas show no or lower expression of HDAC9 (Fisher’s exact test P=0.004; as compared to DLBCL). Classical HL showed frequently low-expression of HDAC9 in the tumor cells. In summary, HDAC9 is frequently expressed in B-cell lymphomas with the highest level of expression found in the most aggressive lymphomas such as DLBCL and MCL. These findings support the biological role of HDAC9 in the pathobiology of aggressive B cell neoplasms. Disclosures No relevant conflicts of interest to declare.

Triple Negative Breast cancer (TNBC) is an aggressive subtype of breast cancer associated with early recurrence and poor prognosis. The treatment options are limited due to lack of expression of common drug targets: estrogen receptor (ER), Progesterone receptor (PR) and Epidermal growth factor receptor 2 (Her2). Epigenetic programs can generate aberrant transcription contributing to TNBC progression; however the dynamic and reversible nature of epigenetic changes offers the possibility to reprogram cancer cells to re-express targets that can render TNBC sensitive to targeted therapies like tamoxifen. Envisioning such ‘epidrugs’, we previously published that targeting PAH2 domain of the master transcriptional scaffold Sin3 by stable expression of 13-mer peptide corresponding to a specific motif called SID (mSin3A interaction domain) disrupts its interaction with a small group of SID-containing transcription factors. This interference reverts the expression of important breast cancer-associated genes and impairs tumor growth in vivo. We have now extended our study towards the evaluation of a cell penetrating SID peptide (pSID) in in vitro and in vivo models to establish parameters for the design of targeted epigenetic therapy for TNBC. pSID co-localizes with Sin3A and interference with PAH2-mediated Sin3A functions by pSID is shown by disruption of Sin3A-MAD1 interactions in Co-IP and Duo-Link assays. pSID treatment in MDA-MB 231 cells results in functional re-expression of CDH1 and ER along with increased H3K4 and decreased H3K27 methylation on their promoters. We also show reduction in the tumorsphere formation by pSID-pretreated MDA-MB-231 cells indicating possible epigenetic reprogramming of tumor initiating stem cells towards a differentiated phenotype. Support to this hypothesis is added by the 50% reduction in tumor growth and re-expression of CDH1 observed in FVB mice injected with pSID-pretreated MMTV-myc cells. Moreover, microarray expression analysis indicates pSID-induced EMT reversal, increased cell adhesion and reduced cell migration. Intriguingly, upon further dissection of the mechanism of epigenetic regulation by pSID we show dissociation of two important chromatin readers/modifiers from the Sin3 complex: histone H3K4Me3/2 demethylase JARID1B and H3K4Me0 binding PHD-like domain containing protein PF1; both with significantly correlated overexpression in invasive breast carcinoma. We also observe loss of recruitment of JARID1B but not Sin3A from the CDH1 promoter. Currently studies are underway to understand the cooperative role between JARID1B and PF1 in potentiating the aberrant transcription regulation by Sin3 at important breast cancer-associated promoters that can be selectively reprogrammed by SID decoys. We believe this selectivity can limit the otherwise adverse affects that may be observed by the use of generic HDAC inhibitors and demethylating agents. Citation Format: Rossitza Christova, Kevin Petrie, Nidhi Bansal, Boris Leibovitch, Louise Howell, Veronica Gil, Ming-Ming Zhou, Edgardo Ariztia, Eduardo Farias, Arthur Zelent, Samuel Waxman. Targeted PF1, JARID1B inhibition induces epigenetic reprogramming in triple negative breast cancer. [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 411. doi:10.1158/1538-7445.AM2014-411

Introduction: Current therapy for acute myeloid leukemia (AML) is inadequate. Treatment of older patients considered unfit for standard induction therapy is particularly challenging (Juliusson et al. Blood 2009). Hypomethylating agents (HMA) are commonly used alternatives for these patients (Fenaux et al. JCO 2010). In the case of azacitidine, complete and partial response rates range between 15-30% (Maurillo et al. Cancer 2012) and the addition of other drugs (e.g. lenalidomide and vorinostat) has been limited by toxicity. In this study, we sought to improve the activity of azacitidine by adding the non-steroidal anti-inflammatory drug (NSAID), sodium salicylate (NSal). NSAIDs exert anti-cancer effects through impaired signal transduction (inhibition of NF-kB and Wnt/B catenin pathways) (Kop et al. Science 1994; Reya et al. Nature 2003), epigenetic modulation and disruption of cellular metabolism (activation of AMPK) (Wang et al. CMLS 2013). In addition, previous reports have demonstrated that NSal combines synergistically with established anti-leukemic agents (daunorubicin) (Klampfer et al. Blood 1999), through down-regulation of the anti-apoptotic protein Mcl-1. Importantly, NSal has no effect on platelet function and a small pilot trial (N=11) confirmed the clinical feasibility of this agent in patients with refractory myeloid neoplasms (Klimek et al. Leukemia Research 2012). In this study, therapeutic plasma concentrations of NSal were safely achieved without excessive NSAID class effects (GI and renal toxicity, bleeding). Here, we report the in vitro effects of NSal combined with azacitidine and suggest a rationale for exploring this combination further in the clinic.

Histone modifications play a crucial role in the regulation of gene expression by activating or inactivating transcription. The Polycomb Group Protein Enhancer of Zeste Homologue 2 (EZH2) mediates trimethylation of histone H3K27, thereby inducing gene silencing. Overexpression of EZH2 has been reported to be associated with metastases and cancer progression in solid tumors like breast cancer or prostate cancer. However, loss of function mutations or deletions of EZH2 occur in myeloid malignancies and T-ALL. These mutations result in a poor prognosis. The aim of this study was to analyze the relevance of histone modifications for therapy resistance in AML. FLT3-ITD positive MV4-11 leukemic cells that were continuously cultured in media containing the kinase inhibitor PKC412 became resistant not only to PKC412 but also to standard chemotherapeutics Cytarabin (AraC) and Daunorubicin. Western blot analysis identified an almost complete loss of H3K27me3 in resistant MV4-11 cells (MV4-11R). This was accompanied by loss of EZH2 protein in the MV4-11R compared to the sensitive MV4-11. To test for acquisition of drug resistance due to reduced H3K27me3 levels, lentiviral knock-down (KD) of EZH2 was performed in the sensitive MV4-11 leading to diminished H3K27me3 levels. Knock-down cells showed resistance to the apoptosis-inducing effects of PKC412 compared to scrambled controls. Furthermore, resistance to standard chemotherapeutics AraC and Daunorubicin could be also observed in MV4-11 KD cells compared to control. To verify whether diminished levels of H3K27me3 can cause a more general, FLT3-ITD-independent drug resistance, knock-down of EZH2 was performed in FLT3-WT AML cell lines HL60, Kasumi-1 and ML-1. Again, this led to resistance to the standard chemotherapeutics AraC and Daunorubicin. In order to investigate the regulation of EZH2 in MV4-11R, promoter methylation and microRNA expression analysis was performed revealing no regulation of EZH2 expression via both mechanisms. Instead, the reduction of EZH2 protein expression was depended on posttranslational mechanisms that could be counteracted by CDK1-inhibitors. CDK1-inhibitors restored EZH2 protein and H3K27 trimethylation levels as well as drug sensitivity. By analyzing EZH2 mRNA expression of 220 primary diagnosed AML patients, a trend towards low EZH2 mRNA expression and poor overall as well as relapse free survival could be demonstrated. Thus, EZH2- and H3K27me3 protein expression were also analyzed by immunohistochemistry in bone marrow biopsies from AML patients (N=126). H3K27me3 and EZH2 protein expression correlated closely (r= 0.9, p<0.001). Low H3K27me3 levels indicated a poor prognosis with significantly decreased overall (median 11.06 vs. 38.6 months, p=0.017), event-free (median 4 vs. 19.9 months, p=0.014) and relapse-free survival (median 10 versus 31.2 months, p=0.038) compared to patients with high H3K27me3 protein expression. Similar findings were obtained for the loss of EZH2 protein. EZH2 low/absent expression was associated with a significantly decreased overall (median 9.6 vs. 47.6 months, p=0.018), event-free (median 4.06 vs. 46 months, p=0.013) and relapse-free survival (median 11.3 versus 55.3 months, p=0.47) compared to patients with high EZH2 protein expression. Taken together, these data indicate that loss of EZH2 expression and reduction of H3K27me3 levels induce widespread therapy resistance in AML and associate with a poor prognosis in AML patients. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Cells present as minimal residual disease (MRD) following frontline therapy drive chemoresistant tumour relapse, thus their detection and targeting is a major priority in cancer therapy. Neuroblastoma (NB) is an aggressive neural crest-derived malignancy of infants and young children. A hallmark of NB is its clinical heterogeneity and the majority of infants with NB have a unique biology that results in spontaneous remission through differentiation in the absence of therapy, despite disease that may be disseminated to the liver, skin, and bone marrow at diagnosis. These infants (with stage 4S disease) have survival rates that exceed 95%. Children with high-risk NB, however, largely characterized by amplification of the MYCN oncogene, often have a disease that is therapy resistant. Although modest improvements in outcome have been achieved as a result of therapy intensification, 5-year event free survival in high-risk patients remains approximately 40-50%. Typically, high-risk neuroblastoma recurs after near complete remission is achieved and the development of effective strategies directed at MRD remains an unmet clinical need. Detection of MRD in neuroblastoma is usually performed using real-time quantitative (RQ)-PCR of neuroblastoma-specific transcripts. Tumor-selective mRNA markers levels are highly dependent on gene expression, which can vary between patients (by a factor of up 1000) and change during treatment. Furthermore, the applicability of a PCR target for MRD detection is also determined by its background expression in hematologic cells. Chromatin conformation patterns integrate the spatial arrangement of the chromatin and build a signature for the physiological status of the cell representing early changes in the genetic and epigenetic regulation. These signatures form the basis for a new technology, Episwitch™, which can identify epigenetic patterns linked with cancer progression and aggressiveness. Results: Based on a screen of human NB cell lines containing amplification of MYCN or expression of high levels of MYCN protein versus NB cell lines lacking expression of MYCN, we have identified a panel of four NB-associated genetic markers: HDM2, PHOX2B, TERT and TH. HDM2 and TERT are direct transcriptional targets of MYCN. This epigenetic signature is currently being validated in primary NB samples. An in vivo proof of concept study utilizing the equivalent mouse epigenetic signature is being performed in a relapse model of MYCN-driven NB. Conclusion: In this study we have developed and tested a novel blood test based on Episwitch™ technology that discriminates MYCN-associated NB. Our data suggest this non-invasive test can be used to monitor remission/relapse status quantitatively with high sensitivity in high-risk MYCN-associated neuroblastoma, thus informing treatment decisions. Citation Format: Kevin Petrie, Megan Field, Zai Ahmad, Mehrnoush Dezfouli, Karen Barker, Magdalena Jeznach, Laura Glass, Albert Hallsworth, Yordan Sbirkov, Howard Womersley, Arthur Zelent, Philip Jordan, Alexandre Akoulitchev, Louis Chesler. A novel epigenetic blood test to monitor minimal residual disease in high-risk neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-87. doi:10.1158/1538-7445.AM2013-LB-87

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The Sin3 A/B adapter proteins function as structural scaffolds for repressor/activator complexes that regulate transcription through the specific association with histone modifying enzymes and a number of transcription factors. Sin3 contains four paired amphipathic α-helices (PAH domains). We have reported earlier that targeted disruption of the PAH2 domain with a SID (Sin3 Interaction Domain) peptide decoy in triple negative (TN) breast cancer cells leads to cytoskeletal reorganization, loss of anchorage independent growth and 3D invasive morphology and decreased cell adhesion and invasion. There is epigenetic reprogramming of silenced genes such as CDH1, ESR1 and RARA which are re-expressed and together contribute to a SID decoy induced switch from basal to a more differentiated luminal phenotype (Farias, et. al., PNAS, 2010, 107:11811-6). Computerized screening coupled with such assays as Duolink, GST pull downs and mammalian two hybrid identified small molecule inhibitors (SMI) that mimic the effects of the SID decoy peptide. SMI inhibit cellular invasion at nanomolar range and in in vivo mouse myc TN breast cancer prolong latency, decrease local invasion and metastasis. Tumors recovered showed evidence of re-expression of E-cadherin and estrogen receptor. Early effects of SID decoy in TN breast cancer cells include inhibition of invasion that is associated with a significant decrease in Src phosphorylation within 2-4hr of treatment. Recovery of phosphorylation after SID decoy washout is coupled with recovered invasion at 24hr. These effects occurred prior to measurable increase in E-cadherin expression, suggesting a non-transcriptional effect. In Drosophila larval breast cancer models with activated Src/Ras, overexpression of SID inhibited (60%) tumor growth in the eye imaginal disc, and was eradicated by the addition of a MEK inhibitor (AZD-6244), indicating a strong synergy between SID and AZD-6244. In the adult fly SID expression greatly inhibited RetMEN2B induced eye tumors (90%). These data demonstrate that SID decoys have a potential to be effective agents in the treatment of TN breast cancer by promoting basal phenotype reversal, inhibiting invasion and metastasis, and could be synergistic with specific inhibitors of signal transduction targets. Moreover, SID decoys can overcome profound oncogenic stimulus such as Ras, Src and Ret suggesting that they have a potential greater role than just in the treatment of TN breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1826. doi:1538-7445.AM2012-1826

Abstract Abstract 3505 Histone Deacetylase 9 (HDAC9) is a class IIa enzyme that, within the hematopoietic system, is preferentially expressed in the B-cell lineage. Previously we have observed that, compared with normal cellular counterparts, expression of HDAC9 appears deregulated in B-lymphoid tumors (Petrie et al., JBC 278: 16059, 2003 and unpublished results). To examine the role of HDAC9 in B-cell transformation we generated a mouse model that constitutively expresses human HDAC9 from early stages of B-cell development under the control of the immunoglobulin heavy chain (IgH) enhancer (Eμ). We demonstrate that from six months of age (6–12 months), Eμ-HDAC9 transgenic mice displayed splenomegaly in a statistically significant fraction of cases (3/17, 18%), which was associated with histopathologic and immunophenotypic evidence of lymphoproliferative disease (LPD) (p<0.0001). Later in life (12 to 23 months of age), 24% (19/78) of Eμ-HDAC9 mice developed a spectrum of mature clonal B-cell lymphoproliferations including monoclonal B-cell lymphocytosis (MBL, 12/78, 15%), splenic marginal zone lymphoma (SMZL, 4/78, 5%) and diffuse large B-cell lymphoma (DLBCL, 3/78, 4%). In contrast, a low background incidence of these malignancies (2%) was observed in control mice (p = 0.0008). Importantly, the HDAC9 gene is located within chromosomal region 7p21, which is a common target of frequent genomic gains in B-cell non-Hodgkin lymphoma (B-NHL) cases and B-NHL cell lines. Consistently, copy number gains in the 7p21.1 chromosomal region were found in 25/79 (32%) primary DLBCL cases analyzed (including one B-NHL patient with a copy number gain only within the 5' end of the HDAC9 gene). Mechanism underlying the role of HDAC9 in lymphomagenesis appears to involve deacetylation of both p53 (leading to deactivation of p53) and BCL6 (leading to activation of BCL6), consistent with a role of a general hypoacetylated state in B-NHL pathogenesis, which is underscored by recently identified inactivating mutations in a number of key histone acetyltransferases. In summary, aberrant expression of HDAC9 leads to the development of B-cell lymphoproliferations, including germinal center (GC) and post-GC lymphomas, showing similarities with those occurring in humans. These findings have relevant therapeutic implications in view of current attempts to use HDAC inhibitors as anti-lymphoma drugs. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 224 During hematopoiesis, all-trans-retinoic acid (ATRA), a natural derivative of vitamin A, has been shown to induce both myelomonocytic progenitor/stem cell differentiation and self-renewal. Although these opposing effects are likely to be partly due to developmental differences, it has been shown that pro- and anti-differentiation effects of ATRA are mediated by distinct retinoic acid receptor isotypes (RARα and RARγ, respectively). With the exception of acute promyelocytic leukemia (APL), ATRA treatment as a single agent has not been successful in other types of acute myeloid leukemia (AML). We have previously hypothesized that one of the underlying reasons for poor response of non-APL AML to ATRA (pan-RAR agonist) is aberrant expression and/or activities of RAR isotypes favoring RARγ and cell growth versus differentiation. Consistently, we have reported that expression of RARα isoforms, particularly ATRA-inducible RARα2, are down-regulated in AML (Blood. 2008; 111:2374). Epigenetic analysis of patient samples revealed that relative to normal CD33+ cells, the loss of RARα2 in AML is associated with a diminution in levels of histone histone H3 lysine 4 dimethylation (H3K4me2) on the ATRA-responsive RARA2 promoter (a modification associated with transcriptional activation). Interestingly, the H3K4me1/me2 demethylase LSD1/KDM1 (AOF2) is highly expressed in AML patients (www.proteinatlas.org). A number of small molecules that target this enzyme (LSD1i) are in development and, collectively, these data predict that the use of LSD1i will facilitate induction of expression of genes that are required for differentiation of AML cells. In this study we used tranylcypromine (TCP, a monoamine oxidase used as an antidepressant and anxiolytic agent in the clinical treatment of mood and anxiety disorders, respectively), which functions a time-dependent, mechanism-based inhibitor of LSD1. Here we show that TCP unlocked the ATRA-driven therapeutic differentiation response in non-APL AML cell lines including the TEX cell line, which is derived from primitive human cord blood cells immortalized by expression of the TLS-ERG oncogene. TEX cells are >90% CD34+, respond poorly to ATRA and mimic features of primary human AML and leukemia initiating cells (Leukemia. 2005; 19:1794). Consistent with this, ATRA/TCP treatment increased differentiation in primary patient samples. ATRA alone had in general only small effects in primary AML samples and TCP showed minimal activity in most cases. Furthermore, shRNA-mediated knockdown of LSD1 confirmed a critical role for this enzyme in blocking the ATRA response in AML cells. The effects of ATRA/TCP on AML cell maturation were paralleled by enhanced induction of genes associated with myelomonocytic differentiation, including direct ATRA targets. LSD1i treatment did not lead to an increase in genome-wide H3K4me2, but did increase H3K4 dimethylation of myelomonocytic differentiation-associated genes. Importantly, treatment with ATRA/TCP dramatically diminished the clonogenic capacity of AML cells in vitro and engraftment of cells derived from AML patients in vivo, suggesting that ATRA/TCP may also target leukemic stem cells. These data strongly suggest that LSD1 may, at least in part, contribute to AML pathogenesis by inhibiting the normal function of ATRA in myelomonocytic development and pave the way for effective differentiation therapy of AML. Disclosures: No relevant conflicts of interest to declare.

Abstract We have previously reported that site-specific disruption of the Sin3 complex results in targeted epigenetic reprogramming and differentiation in TN breast cancer (Farias et al., PNAS 2010, 107:11811-6). The Sin3 A/B are multidomain adapter proteins part of a multisubunit corepressor scaffold that regulates transcription via the recruitement of HDAC and BP2/JARID1 A. Sin3 (A/B) contain four paired amphipathic α-helices known as PAH domains, a central HID (HDAC interaction domain) also serves as docking platform for a variety of corepressors, a C-terminal domain is a highly conserved region. We have targeted the disruption of the PAH2 domain binding to SID (mSin3A interaction domain) containing transcription factors such as MAD1, REST, KLF-9, -10, -11, -13 and -16, using a peptide decoy containing the SID motif. These studies have been carried out in several breast cancer cell lines including 5 TN, 2 estrogen receptor positive (ER+) and a non-transformed mammary cell line. Transfection with a plasmid expressing minimal SID or treatment with a 13 amino-acid peptide (SID peptide) disrupts transcription factor binding to the PAH2 domain resulting in extreme changes in morphology, cytoskeletal organization, adhesion, decreased extracellular proteolytic activity (MMP-9 and uPA), loss of invasive capacity and anchorage independent growth. There was re-expression of functional E-cadherin, estrogen and retinoic acid receptors. Effects in vivo xenograft also included inhibition of tumor growth and metastasis in FVB mice. 3D cultures in Matrigel have revealed a SID decoy dependent switch from a basal to a more luminal phenotype. These effects of SID decoy occurred in TN but not in ER+ or non-transformed breast cancer cell lines. Taken together, these results support the hypothesis in which SID decoy interference with PAH2 domain binding can revert Epithelial/Mesenchymal Transition in TN breast cancer cells and results in a less malignant phenotype. Fourteen candidate small molecule inhibitors, from a computerized screen of 115,000 compounds, were tested in a mammalian two hybrid assay and some of them have been tested in cellular invasion, proliferation and morphogenesis assays. One of them, compound 14 (C14) has shown no toxicity, induces E-cadherin expression and is effective in inhibiting Matrigel invasion of MDA-MB-231 at 100 nM. 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 2826. doi:10.1158/1538-7445.AM2011-2826

Abstract Our recent work in understanding silenced retinoic acid response genes in breast cancer led us to explore the role of transcription repressor complexes in gene silencing in breast cancer. To this end we constructed a set of tagged vectors that contain a specific MAD1 motif called SID (mSin3A interaction domain), which binds with high affinity to block the function of the Sin3. Sin3A/B serve as multisubunit co-repressor scaffold protein that regulate gene transcription by recruiting histone deacetylase and histone demethylase activities to sequence-specific transcriptional repressors which are aberrant in breast cancer. The PAH2 domain of Sin3A/B binds with high affinity to a small number of transcription factors, and offers a more specific epigenetic target which contributes to the development of breast cancer. PAH-2 domain a specific component of a transcriptional repressor complex that plays an important role in modulating a small number of transcription factors containing the Sin3 PAH-2 interaction domain (SID). Here we demonstrated that in both human and mouse breast cancer cells, the targeted disruption of Sin3 function by introduction with their partners by the expression of SID transcript or peptide decoy interfered with PAH2 binding to SID-containing partner proteins as measured by co-immunoprecipitation and mammalian two-hybrid assays, reverteds the silencing of several genes involved in cell growth and differentiation. We observed that the SID decoy induced clear signs of differentiation in both human and mouse breast cancer cellsIn particular, the which include theSID decoys led to acinar morphogenesis in 3D cultures, increased adherence to collagen type-IV and laminin, reduced invasive phenotype and impaired tumor growth in vivo (>75%). This was associated with epigenetic reprogramming characterized by a marked increase in H3K4 2/3 methylation and a modest increase in H3 acetylation in the promoter region, promoter DNA demethylation and re-expression of the important breast cancer-associated silenced genes encoding E-cadherin, and, estrogen receptor α (ERα) and retinoic acid receptor β (RARβ)and impairment in tumor growth in vivo. There was increased expression of E-cadherin, CRBP1 and p27 known RAR response genes. The re-expression of ERα and RARβ in the “triple negative” MDA-MB-231 breast cancer cell line is functional since there was significant growth inhibition by tamoxifen after stimulation with 17b-estradiol and RAR activation by atRA and AM580. Therefore, the development of small molecules that mimic the 13 amino acid SID peptide and block interactions between PAH2 and SID-containing proteins This offers a new novel approach for treating this type of breast cancer and may also provide wider therapeutic implicationstriple negative breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 572.

Sin3A/B is a master transcriptional scaffold and corepressor that plays an essential role in the regulation of gene transcription and maintenance of chromatin structure, and its inappropriate recruitment has been associated with aberrant gene silencing in cancer. Sin3A/B are highly related, large, multidomian proteins that interact with a wide variety of transcription factors and corepressor components, and we examined whether disruption of the function of a specific domain could lead to epigenetic reprogramming and derepression of specific subsets of genes. To this end, we selected the Sin3A/B-paired amphipathic α-helices (PAH2) domain based on its established role in mediating the effects of a relatively small number of transcription factors containing a PAH2-binding motif known as the Sin3 interaction domain (SID). Here, we show that in both human and mouse breast cancer cells, the targeted disruption of Sin3 function by introduction of a SID decoy that interferes with PAH2 binding to SID-containing partner proteins reverted the silencing of genes involved in cell growth and differentiation. In particular, the SID decoy led to epigenetic reprogramming and reexpression of the important breast cancer-associated silenced genes encoding E-cadherin, estrogen receptor α, and retinoic acid receptor β and impaired tumor growth in vivo. Interestingly, the SID decoy was effective in the triple-negative M.D. Anderson-Metastatic Breast-231 (MDA-MB-231) breast cancer cell line, restoring sensitivity to 17β-estradiol, tamoxifen, and retinoids. Therefore, the development of small molecules that can block interactions between PAH2 and SID-containing proteins offers a targeted epigenetic approach for treating this type of breast cancer that may also have wider therapeutic implications.