Looking into the Eyes-In Vitro Models for Ocular Research.

Animal research undoubtedly provides scientists with virtually unlimited data but inflicts pain and suffering on animals. Currently, legislators and scientists alike are promoting alternative in vitro approaches allowing for an accurate evaluation of processes occurring in the body without animal sacrifice. Historically, one of the most infamous animal tests is the Draize test, mainly performed on rabbits. Even though this test was considered the gold standard for around 50 years, the Draize test fails to mimic human response mainly due to human and rabbit eye physiological differences. Therefore, many alternative assays were developed to evaluate ocular toxicity and drug effectiveness accurately. Here we review recent achievements in tissue engineering of in vitro 2D, 2.5D, 3D, organoid and organ-on-chip ocular models, as well as in vivo and ex vivo models in terms of their advantages and limitations.

Suppression of MYC by PI3K/AKT/mTOR pathway inhibition in combination with all-trans retinoic acid treatment for therapeutic gain in acute myeloid leukaemia.

Aberrant activity of the phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR [PAM]) pathway, as well as suppressed retinoic acid signalling, contribute to enhanced proliferation and the differentiation blockade of immature myeloid cells in acute myeloid leukaemia (AML). Inhibition of the PAM pathway was shown to affect especially mixed-lineage leukaemia-rearranged AML. Here, we sought to test a combined strategy using small molecule inhibitors against members of the PAM signalling pathway in conjunction with all-trans retinoic acid (ATRA) to target a larger group of different AML subtypes. We find that ATRA treatment in combination with inhibition of PI3K (ZSTK474), mTOR (WYE132) or PI3K/mTOR (BEZ235, dactolisib) drastically reduces protein levels of the proto-oncogene MYC. In combination with BEZ235, ATRA treatment led to almost complete eradication of cellular MYC, G1 arrest, loss of clonal capacity and terminal granulocytic differentiation. We demonstrate that PAM inhibitor/ATRA treatment targets MYC via independent mechanisms. While inhibition of the PAM pathway causes MYC phosphorylation at threonine 58 via glycogen synthase kinase 3 beta and subsequent degradation, ATRA reduces its expression. Here, we present an approach using a combination of known drugs to synergistically reduce aberrant MYC levels, thereby effectively blocking proliferation and enabling differentiation in various AML subtypes.

Clinical Responsiveness to All-trans Retinoic Acid Is Potentiated by LSD1 Inhibition and Associated with a Quiescent Transcriptome in Myeloid Malignancies.

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.

Retinoic Acid and Its Derivatives in Skin.

The retinoids are a group of compounds including vitamin A and its active metabolite all-trans-retinoic acid (ATRA). Retinoids regulate a variety of physiological functions in multiple organ systems, are essential for normal immune competence, and are involved in the regulation of cell growth and differentiation. Vitamin A derivatives have held promise in cancer treatment and ATRA is used in differentiation therapy of acute promyelocytic leukemia (APL). ATRA and other retinoids have also been successfully applied in a variety of dermatological conditions such as skin cancer, psoriasis, acne, and ichthyosis. Moreover, modulation of retinoic acid receptors and retinoid X (or rexinoid) receptors function may affect dermal cells. The studies using complex genetic models with various combinations of retinoic acid receptors (RARs) and retinoid X (or rexinoid) receptors (RXRs) indicate that retinoic acid and its derivatives have therapeutic potential for a variety of serious dermatological disorders including some malignant conditions. Here, we provide a synopsis of the main advances in understanding the role of ATRA and its receptors in dermatology.

Correction: The acetyltransferase GCN5 maintains ATRA-resistance in non-APL AML.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The acetyltransferase GCN5 maintains ATRA-resistance in non-APL AML.

To date, only one subtype of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL) can be effectively treated by differentiation therapy utilizing all-trans retinoic acid (ATRA). Non-APL AMLs are resistant to ATRA. Here we demonstrate that the acetyltransferase GCN5 contributes to ATRA resistance in non-APL AML via aberrant acetylation of histone 3 lysine 9 (H3K9ac) residues maintaining the expression of stemness and leukemia associated genes. We show that inhibition of GCN5 unlocks an ATRA-driven therapeutic response. This response is potentiated by coinhibition of the lysine demethylase LSD1, leading to differentiation in most non-APL AML. Induction of differentiation was not correlated to a specific AML subtype, cytogenetic, or mutational status. Our study shows a previously uncharacterized role of GCN5 in maintaining the immature state of leukemic blasts and identifies GCN5 as a therapeutic target in AML. The high efficacy of the combined epigenetic treatment with GCN5 and LSD1 inhibitors may enable the use of ATRA for differentiation therapy of non-APL AML. Furthermore, it supports a strategy of combined targeting of epigenetic factors to improve treatment, a concept potentially applicable for a broad range of malignancies.

Ex-vivo sensitivity profiling to guide clinical decision making in acute myeloid leukemia: A pilot study.

A precision medicine approach is appealing for use in AML due to ease of access to tumor samples and the significant variability in the patients' response to treatment. Attempts to establish a precision medicine platform for AML, however, have been unsuccessful, at least in part due to the use of small compound panels and having relatively slow turn over rates, which restricts the scope of treatment and delays its onset. For this pilot study, we evaluated a cohort of 12 patients with refractory AML using an ex vivo drug sensitivity testing (DST) platform. Purified AML blasts were screened with a panel of 215 FDA-approved compounds and treatment response was evaluated after 72h of exposure. Drug sensitivity scoring was reported to the treating physician, and patients were then treated with either DST- or non-DST guided therapy. We observed survival benefit of DST-guided therapy as compared to the survival of patients treated according to physician recommendation. Three out of four DST-treated patients displayed treatment response, while all of the non-DST-guided patients progressed during treatment. DST rapidly and effectively provides personalized treatment recommendations for patients with refractory AML.

Targeted interference of SIN3A-TGIF1 function by SID decoy treatment inhibits Wnt signaling and invasion in triple negative breast cancer cells.

Cancer cell invasion is an obligatory step for metastatic dissemination that contributes to rapid relapse and a poorer survival in triple negative breast cancer (TNBC) patients. Development of novel therapeutic strategies to block tumor invasion is an unmet need in the treatment of cancer. We reported that the selective inhibition of the PAH2 domain of SIN3A protein function markedly suppressed metastatic dissemination to the lungs in TNBC xenograft bearing mice. Here, we show that TNBC cell lines treated with Sin3 interaction domain (SID) decoy peptides that bind to PAH2 display a strong in vitro inhibition of transwell invasion. This is accompanied by actin cytoskeleton reorganization with increased cortical actin deposition and downregulation of known Wnt target genes that are associated with epithelial to mesenchymal transition (EMT) and cancer cell invasion. Wnt pathway inhibition by SID decoy peptide was confirmed by decreased Wnt reporter activity and altered cytoplasmic localization of nuclear ß-catenin. TGIF1, a transcription factor that modulates Wnt signaling and known to interact with the PAH2 domain of SIN3A, can be dissociated from the SIN3A complex by SID decoys. TGIF1 knockdown inhibits WNT target genes and in vitro cell invasion suggesting that TGIF1 might be a key target of the SID decoys to block tumor invasion. Taken together, targeting SIN3 function using SID decoys is a novel strategy to reverse invasion and the EMT program in TNBC translating into the inhibition of metastasis dissemination and eradication of residual disease.

A Case of AML Characterized by a Novel t(4;15)(q31;q22) Translocation That Confers a Growth-Stimulatory Response to Retinoid-Based Therapy.

Here we report the case of a 30-year-old woman with relapsed acute myeloid leukemia (AML) who was treated with all-trans retinoic acid (ATRA) as part of investigational therapy (NCT02273102). The patient died from rapid disease progression following eight days of continuous treatment with ATRA. Karyotype analysis and RNA-Seq revealed the presence of a novel t(4;15)(q31;q22) reciprocal translocation involving the TMEM154 and RASGRF1 genes. Analysis of primary cells from the patient revealed the expression of TMEM154-RASGRF1 mRNA and the resulting fusion protein, but no expression of the reciprocal RASGRF1-TMEM154 fusion. Consistent with the response of the patient to ATRA therapy, we observed a rapid proliferation of t(4;15) primary cells following ATRA treatment ex vivo. Preliminary characterization of the retinoid response of t(4;15) AML revealed that in stark contrast to non-t(4;15) AML, these cells proliferate in response to specific agonists of RARα and RARγ. Furthermore, we observed an increase in the levels of nuclear RARγ upon ATRA treatment. In summary, the identification of the novel t(4;15)(q31;q22) reciprocal translocation opens new avenues in the study of retinoid resistance and provides potential for a new biomarker for therapy of AML.

Semi-Quantitative Mass Spectrometry in AML Cells Identifies New Non-Genomic Targets of the EZH2 Methyltransferase.

Alterations to the gene encoding the EZH2 (KMT6A) methyltransferase, including both gain-of-function and loss-of-function, have been linked to a variety of haematological malignancies and solid tumours, suggesting a complex, context-dependent role of this methyltransferase. The successful implementation of molecularly targeted therapies against EZH2 requires a greater understanding of the potential mechanisms by which EZH2 contributes to cancer. One aspect of this effort is the mapping of EZH2 partner proteins and cellular targets. To this end we performed affinity-purification mass spectrometry in the FAB-M2 HL-60 acute myeloid leukaemia (AML) cell line before and after all-trans retinoic acid-induced differentiation. These studies identified new EZH2 interaction partners and potential non-histone substrates for EZH2-mediated methylation. Our results suggest that EZH2 is involved in the regulation of translation through interactions with a number of RNA binding proteins and by methylating key components of protein synthesis such as eEF1A1. Given that deregulated mRNA translation is a frequent feature of cancer and that eEF1A1 is highly expressed in many human tumours, these findings present new possibilities for the therapeutic targeting of EZH2 in AML.

Loss of the histone methyltransferase EZH2 induces resistance to multiple drugs in acute myeloid leukemia.

In acute myeloid leukemia (AML), therapy resistance frequently occurs, leading to high mortality among patients. However, the mechanisms that render leukemic cells drug resistant remain largely undefined. Here, we identified loss of the histone methyltransferase EZH2 and subsequent reduction of histone H3K27 trimethylation as a novel pathway of acquired resistance to tyrosine kinase inhibitors (TKIs) and cytotoxic drugs in AML. Low EZH2 protein levels correlated with poor prognosis in AML patients. Suppression of EZH2 protein expression induced chemoresistance of AML cell lines and primary cells in vitro and in vivo. Low EZH2 levels resulted in derepression of HOX genes, and knockdown of HOXB7 and HOXA9 in the resistant cells was sufficient to improve sensitivity to TKIs and cytotoxic drugs. The endogenous loss of EZH2 expression in resistant cells and primary blasts from a subset of relapsed AML patients resulted from enhanced CDK1-dependent phosphorylation of EZH2 at Thr487. This interaction was stabilized by heat shock protein 90 (HSP90) and followed by proteasomal degradation of EZH2 in drug-resistant cells. Accordingly, inhibitors of HSP90, CDK1 and the proteasome prevented EZH2 degradation, decreased HOX gene expression and restored drug sensitivity. Finally, patients with reduced EZH2 levels at progression to standard therapy responded to the combination of bortezomib and cytarabine, concomitant with the re-establishment of EZH2 expression and blast clearance. These data suggest restoration of EZH2 protein as a viable approach to overcome treatment resistance in this AML patient population.

Deregulated expression of HDAC9 in B cells promotes development of lymphoproliferative disease and lymphoma in mice.

Histone deacetylase 9 (HDAC9) is expressed in B cells, and its overexpression has been observed in B-lymphoproliferative disorders, including B-cell non-Hodgkin lymphoma (B-NHL). We examined HDAC9 protein expression and copy number alterations in primary B-NHL samples, identifying high HDAC9 expression among various lymphoma entities and HDAC9 copy number gains in 50% of diffuse large B-cell lymphoma (DLBCL). To study the role of HDAC9 in lymphomagenesis, we generated a genetically engineered mouse (GEM) model that constitutively expressed an HDAC9 transgene throughout B-cell development under the control of the immunoglobulin heavy chain (IgH) enhancer (Eµ). Here, we report that the Eµ-HDAC9 GEM model develops splenic marginal zone lymphoma and lymphoproliferative disease (LPD) with progression towards aggressive DLBCL, with gene expression profiling supporting a germinal center cell origin, as is also seen in human B-NHL tumors. Analysis of Eµ-HDAC9 tumors suggested that HDAC9 might contribute to lymphomagenesis by altering pathways involved in growth and survival, as well as modulating BCL6 activity and p53 tumor suppressor function. Epigenetic modifications play an important role in the germinal center response, and deregulation of the B-cell epigenome as a consequence of mutations and other genomic aberrations are being increasingly recognized as important steps in the pathogenesis of a variety of B-cell lymphomas. A thorough mechanistic understanding of these alterations will inform the use of targeted therapies for these malignancies. These findings strongly suggest a role for HDAC9 in B-NHL and establish a novel GEM model for the study of lymphomagenesis and, potentially, preclinical testing of therapeutic approaches based on histone deacetylase inhibitors.

Blocking the PAH2 domain of Sin3A inhibits tumorigenesis and confers retinoid sensitivity in triple negative breast cancer.

Triple negative breast cancer (TNBC) frequently relapses locally, regionally or as systemic metastases. Development of targeted therapy that offers significant survival benefit in TNBC is an unmet clinical need. We have previously reported that blocking interactions between PAH2 domain of chromatin regulator Sin3A and the Sin3 interaction domain (SID) containing proteins by SID decoys result in EMT reversal, and re-expression of genes associated with differentiation. Here we report a novel and therapeutically relevant combinatorial use of SID decoys. SID decoys activate RARα/ß pathways that are enhanced in combination with RARα-selective agonist AM80 to induce morphogenesis and inhibit tumorsphere formation. These findings correlate with inhibition of mammary hyperplasia and a significant increase in tumor-free survival in MMTV-Myc oncomice treated with a small molecule mimetic of SID (C16). Further, in two well-established mouse TNBC models we show that treatment with C16-AM80 combination has marked anti-tumor effects, prevents lung metastases and seeding of tumor cells to bone marrow. This correlated to a remarkable 100% increase in disease-free survival with a possibility of "cure" in mice bearing a TNBC-like tumor. Targeting Sin3A by C16 alone or in combination with AM80 may thus be a promising adjuvant therapy for treating or preventing metastatic TNBC.

Targeting the SIN3A-PF1 interaction inhibits epithelial to mesenchymal transition and maintenance of a stem cell phenotype in triple negative breast cancer.

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.

Post transcriptional control of the epigenetic stem cell regulator PLZF by sirtuin and HDAC deacetylases.

BACKGROUND: The transcriptional repressor promyelocytic leukemia zinc finger protein (PLZF) is critical for the regulation of normal stem cells maintenance by establishing specific epigenetic landscape. We have previously shown that CBP/p300 acetyltransferase induces PLZF acetylation in order to increase its deoxynucleotidic acid (DNA) binding activity and to enhance its epigenetic function (repression of PLZF target genes). However, how PLZF is inactivated is not yet understood. RESULTS: In this study, we demonstrate that PLZF is deacetylated by both histone deacetylase 3 and the NAD+ dependent deacetylase silent mating type information regulation 2 homolog 1 (SIRT1). Unlike other PLZF-interacting deacetylases, these two proteins interact with the zinc finger domain of PLZF, where the activating CBP/p300 acetylation site was previously described, inducing deacetylation of lysines 647/650/653. Overexpression of histone deacetylase 3 (HDAC3) and SIRT1 is associated with loss of PLZF DNA binding activity and decreases PLZF transcriptional repression. As a result, the chromatin status of the promoters of PLZF target genes, involved in oncogenesis, shift from a heterochromatin to an open euchromatin environment leading to gene expression even in the presence of PLZF. CONCLUSIONS: Consequently, SIRT1 and HDAC3 mediated-PLZF deacetylation provides for rapid control and fine-tuning of PLZF activity through post-transcriptional modification to regulate gene expression and cellular homeostasis.

Selective Inhibition of SIN3 Corepressor with Avermectins as a Novel Therapeutic Strategy in Triple-Negative Breast Cancer.

Triple-negative breast cancers (TNBC) lacking estrogen, progesterone, and HER2 receptors account for 10% to 20% of breast cancer and are indicative of poor prognosis. The development of effective treatment strategies therefore represents a pressing unmet clinical need. We previously identified a molecularly targeted approach to target aberrant epigenetics of TNBC using a peptide corresponding to the SIN3 interaction domain (SID) of MAD. SID peptide selectively blocked binding of SID-containing proteins to the paired α-helix (PAH2) domain of SIN3, resulting in epigenetic and transcriptional modulation of genes associated with epithelial-mesenchymal transition (EMT). To find small molecule inhibitor (SMI) mimetics of SID peptide, we performed an in silico screen for PAH2 domain-binding compounds. This led to the identification of the avermectin macrocyclic lactone derivatives selamectin and ivermectin (Mectizan) as candidate compounds. Both selamectin and ivermectin phenocopied the effects of SID peptide to block SIN3-PAH2 interaction with MAD, induce expression of CDH1 and ESR1, and restore tamoxifen sensitivity in MDA-MB-231 human and MMTV-Myc mouse TNBC cells in vitro. Treatment with selamectin or ivermectin led to transcriptional modulation of genes associated with EMT and maintenance of a cancer stem cell phenotype in TNBC cells. This resulted in impairment of clonogenic self-renewal in vitro and inhibition of tumor growth and metastasis in vivo. Underlining the potential of avermectins in TNBC, pathway analysis revealed that selamectin also modulated the expression of therapeutically targetable genes. Consistent with this, an unbiased drug screen in TNBC cells identified selamectin-induced sensitization to a number of drugs, including those targeting modulated genes.

Histone deacetylase 9 promotes angiogenesis by targeting the antiangiogenic microRNA-17-92 cluster in endothelial cells.

OBJECTIVE: Histone deacetylases (HDACs) modulate gene expression by deacetylation of histone and nonhistone proteins. Several HDACs control angiogenesis, but the role of HDAC9 is unclear. METHODS AND RESULTS: Here, we analyzed the function of HDAC9 in angiogenesis and its involvement in regulating microRNAs. In vitro, silencing of HDAC9 reduces endothelial cell tube formation and sprouting. Furthermore, HDAC9 silencing decreases vessel formation in a spheroid-based Matrigel plug assay in mice and disturbs vascular patterning in zebrafish embryos. Genetic deletion of HDAC9 reduces retinal vessel outgrowth and impairs blood flow recovery after hindlimb ischemia. Consistently, overexpression of HDAC9 increases endothelial cell sprouting, whereas mutant constructs lacking the catalytic domain, the nuclear localization sequence, or sumoylation site show no effect. To determine the mechanism underlying the proangiogenic effect of HDAC9, we measured the expression of the microRNA (miR)-17-92 cluster, which is known for its antiangiogenic activity. We demonstrate that silencing of HDAC9 in endothelial cells increases the expression of miR-17-92. Inhibition of miR-17-20a rescues the sprouting defects induced by HDAC9 silencing in vitro and blocking miR-17 expression partially reverses the disturbed vascular patterning of HDAC9 knockdown in zebrafish embryos. CONCLUSIONS: We found that HDAC9 promotes angiogenesis and transcriptionally represses the miR-17-92 cluster.

Inhibition of the LSD1 (KDM1A) demethylase reactivates the all-trans-retinoic acid differentiation pathway in acute myeloid leukemia.

Acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML), characterized by the t(15;17)-associated PML-RARA fusion, has been successfully treated with therapy utilizing all-trans-retinoic acid (ATRA) to differentiate leukemic blasts. However, among patients with non-APL AML, ATRA-based treatment has not been effective. Here we show that, through epigenetic reprogramming, inhibitors of lysine-specific demethylase 1 (LSD1, also called KDM1A), including tranylcypromine (TCP), unlocked the ATRA-driven therapeutic response in non-APL AML. LSD1 inhibition did not lead to a large-scale increase in histone 3 Lys4 dimethylation (H3K4(me2)) across the genome, but it did increase H3K4(me2) and expression of myeloid-differentiation-associated genes. Notably, treatment with ATRA plus TCP markedly diminished the engraftment of primary human AML cells in vivo in nonobese diabetic (NOD)-severe combined immunodeficient (SCID) mice, suggesting that ATRA in combination with TCP may target leukemia-initiating cells. Furthermore, initiation of ATRA plus TCP treatment 15 d after engraftment of human AML cells in NOD-SCID γ (with interleukin-2 (IL-2) receptor γ chain deficiency) mice also revealed the ATRA plus TCP drug combination to have a potent anti-leukemic effect that was superior to treatment with either drug alone. These data identify LSD1 as a therapeutic target and strongly suggest that it may contribute to AML pathogenesis by inhibiting the normal pro-differentiative function of ATRA, paving the way for new combinatorial therapies for AML.

Histone deacetylases 9 and 10 are required for homologous recombination.

We tested the role of histone deacetylases (HDACs) in the homologous recombination process. A tissue-culture based homology-directed repair assay was used in which repair of a double-stranded break by homologous recombination results in gene conversion of an inactive GFP allele to an active GFP gene. Our rationale was that hyperacetylation caused by HDAC inhibitor treatment would increase chromatin accessibility to repair factors, thereby increasing homologous recombination. Contrary to expectation, treatment of cells with the inhibitors significantly reduced homologous recombination activity. Using RNA interference to deplete each HDAC, we found that depletion of either HDAC9 or HDAC10 specifically inhibited homologous recombination. By assaying for sensitivity of cells to the interstrand cross-linker mitomycin C, we found that treatment of cells with HDAC inhibitors or depletion of HDAC9 or HDAC10 resulted in increased sensitivity to mitomycin C. Our data reveal an unanticipated function of HDAC9 and HDAC10 in the homologous recombination process.

Interference with Sin3 function induces epigenetic reprogramming and differentiation in breast cancer cells.

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 alpha-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 alpha, and retinoic acid receptor beta 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 17beta-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.