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Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including “sawtooth” patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes. Electronic supplementary material The online version of this article (doi:10.1007/s10549-015-3468-2) contains supplementary material, which is available to authorized users.

Aberrations in histone acetyltransferases and histone deacetylases (HDAC) have been associated with tumor development and tumor progression. Pharmacological interference with histone acetylation and methylation has shown to be promising for the treatment and prevention of cancer. Several structurally and functionally different HDAC inhibitors have been developed as a novel modality to reverse aberrant epigenetic changes in cancer cells. With a growing body of literature, there is ample preclinical rationale to test these drugs in various stages and settings of cancer, either as single agents or in rationally designed combinations as a therapeutic strategy in hematological as well as solid tumor malignancies. The first HDAC inhibitor vorinostat (Zolinza) was approved for the treatment of patients with cutaneous T-cell lymphoma. HDAC inhibitors have also demonstrated great promise in treating a variety of noncancer diseases, including epilepsy, cystic fibrosis, spinal muscular atrophy, and human immunodeficiency virus infection.

Assessment of the functional consequences of variants near splice sites is a major challenge in the diagnostic laboratory. To address this issue, we created expression minigenes (EMGs) to determine the RNA and protein products generated by splice site variants (n = 10) implicated in cystic fibrosis (CF). Experimental results were compared with the splicing predictions of eight in silico tools. EMGs containing the full-length Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) coding sequence and flanking intron sequences generated wild-type transcript and fully processed protein in Human Embryonic Kidney (HEK293) and CF bronchial epithelial (CFBE41o-) cells. Quantification of variant induced aberrant mRNA isoforms was concordant using fragment analysis and pyrosequencing. The splicing patterns of c.1585−1G>A and c.2657+5G>A were comparable to those reported in primary cells from individuals bearing these variants. Bioinformatics predictions were consistent with experimental results for 9/10 variants (MES), 8/10 variants (NNSplice), and 7/10 variants (SSAT and Sroogle). Programs that estimate the consequences of mis-splicing predicted 11/16 (HSF and ASSEDA) and 10/16 (Fsplice and SplicePort) experimentally observed mRNA isoforms. EMGs provide a robust experimental approach for clinical interpretation of splice site variants and refinement of in silico tools.

Melanocortin-3 receptor (MC3R) is a canonical MSH receptor that plays an essential role in energy homeostasis. Variants in MC3R have been implicated in obesity in humans and mice. However, interpretation of the functional consequences of these variants is challenging because the translational start site of MC3R is unclear. Using 5′ rapid amplification of cDNA ends, we discovered a novel upstream exon that extends the length of the 5′ untranslated region (UTR) in MC3R without changing the open-reading frame. The full-length 5′ UTR directs utilization of an evolutionarily conserved second in-frame ATG as the primary translation start site. MC3R synthesized from the second ATG is localized to apical membranes of polarized Madin-Darby canine kidney cells, consistent with its function as a cell surface mediator of melanocortin signaling. Expression of MC3R causes relocalization of melanocortin receptor accessory protein 2, an accessory factor for melanocortin-2 receptor, to the apical membrane, coincident with the location of MC3R. In contrast, protein synthesized from MC3R cDNAs lacking the 5′ UTR displayed diffuse cytosolic distribution and has no effect on the distribution of melanocortin receptor accessory protein 2. Our findings demonstrate that a previously unannotated 5′ exon directs translation of MC3R protein that localizes to apical membranes of polarized cells. Together, our work provides insight on the structure of human MC3R and reveals a new pathway for regulation of energy metabolism.

Background Treatment options for sarcoma are limited. Histone deacetylase inhibitors increase the efficacy of topoisomerase II inhibitors by promoting access to chromatin and by down-regulating DNA repair. Thus, combined panobinostat and epirubicin therapy was evaluated to treat refractory sarcoma. Patients and methods Patients with advanced solid tumors were enrolled in a 3 + 3 dose-escalation phase I trial of panobinostat given on days 1, 3, and 5 followed by 75 mg/m2 of epirubicin on day 5 in 21-day cycles, with a dose expansion at maximum tolerated dose (MTD) in 20 sarcoma patients. Peripheral blood mononucleocyte histone acetylation was also evaluated. Results Forty patients received 20–60 mg panobinostat. Dose-limiting toxicities included thrombocytopenia, febrile neutropenia, and fatigue at 60 mg, defining a panobinostat MTD at 50 mg. Four responses were seen in 37 assessable patients, all after progression on prior topoisomerase II inhibitors. For those with sarcoma, 12 of 20 derived clinical benefit (1 partial response and 11 stable disease, median overall survival 8.3 months), including 8 of 14 previously progressed on topoisomerase II therapy. Treatment benefits correlated with increased histone acetylation and decreased neutrophil count on day 5. Conclusions Panobinostat and epirubicin treatment is well tolerated and may reverse anthracycline resistance. Changes in histone acetylation and associated decrease in neutrophil count correlated with clinical benefit and warrant investigation as predictive biomarkers. Clinical trial This trial is registered at www.Clinicaltrials.gov , Identifier: NCT00878904.

Understanding the contribution of dysregulated gene silencing to epigenomic alterations in cancer development provides the rationale for the use of epigenetic modulators, such as histone deacetylase (HDAC) inhibitors, in cancer therapy. HDAC inhibitors have been approved as single agents for cutaneous and peripheral T-cell lymphoma and have shown promising activity in reversing therapy resistance in other tumor types. The effects of HDAC inhibitors on immune modulation have created a recent interest in their potential role in immunotherapy. This review describes the current understanding on integrating HDAC inhibitors into various immunotherapeutic approaches, such as cancer vaccines, adoptive T-cell transfer and immune checkpoint inhibitors. Furthermore, it summarizes promising treatment strategies in epigenetic immune priming from clinical trials that are currently underway.

Background: Breast cancer is one of the most common diseases, second only to lung cancer as the leading cause of cancer death in women. In particular hormone sensitive metastatic breast cancer (MBC) and triple-negative breast cancer (TNBC) remains a therapeutic challenge as resistance develops in almost all patients. Despite aggressive treatment strategies, survival is poor due to the development of resistance, prompting the need for novel approaches. Here, we characterize the molecular mechanisms underlying epigenetic modifiers and in particular histone deacetylase inhibitors (HDACi) as priming modulators of immunotherapy, with a specific focus on TNBC and hormone resistant breast cancers. HDACi represent a new class of anticancer agents that can reverse hormone therapy resistance, resulting in prolonged anti-tumor responses in patients. In addition to their effects on estrogen receptor (ER) signaling, HDACi can also influence immune cell function, including but not limited to modulation of Foxp3+ regulatory T-cells (Tregs), tumor-infiltrating lymphocyte (TILs) composition, as well as induction of the co-inhibitory receptors PD-L1 and PD-1. Under normal conditions the PD-1/PD-L1 pathway down-regulates cytotoxic T-cell activity to maintain immune homeostasis. Cancer cells exploit this pathway in the tumor microenvironment to suppress cytotoxic T-cell activation, significantly diminishing the anti-tumor immune response. In breast cancer, PD-L1 expression is less frequent and mainly found in TNBC, HER2+, ER- and PR- tumors. Increased PD-L1 expression correlates with increased TILs and these criteria together are indicative of improved response rates in breast cancer patients. The PD-1/PD-L1 pathway represents one of the primary immunosuppressive drivers in multiple types of cancer. Thus, inhibiting PD-1/PD-L1 interactions may prevent T-cell suppression and reactivate immunosurveillance mechanisms necessary for tumor cell eradication. Methods and Results: Evaluation of basal PD-L1 expression in a range of human and mouse breast cancer cell lines by western blotting and real-time PCR identified TNBC and HER2+ cells as the highest expressing cells. Testing different epigenetic modifiers, we found that HDACi were able to up-regulate PD-L1 mRNA and protein in a time-dependent manner up to 72 hours. This was a direct transcriptional effect induced by HDACi and was confirmed even in tamoxifen resistant breast cancer cells, characterized by increased basal expression of PD-L1 as compared to the parental cells. To define the role of epigenetic priming in promoting immune cell activation, we co-cultured tumor cells and human peripheral blood mononuclear cell (PBMCs) and performed comprehensive immunophenotyping by flow cytometry. HDACi were able to up-regulate PD-L1 on tumor cells independent of PBMCs, while exhibiting a selective decrease in the frequency of immunosuppressive Tregs. Conclusion: Our data demonstrate that the combination of HDACi with immune checkpoint inhibitors represents a novel therapeutic anti-tumor strategy and warrants further clinical evaluation for the treatment of TNBC and hormone resistant breast cancer. Citation Format: Manuela Terranova Barberio, Scott Thomas, Niwa H. Ali, Jeenah Park, Michael D. Rosenblum, Alfredo Budillon, Pamela N. Munster. HDAC inhibitors modulate immune checkpoint blockade in breast cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B111.

Introduction The emergence of hormone therapy resistance, despite continued expression of the estrogen receptor (ER), is a major challenge to curing breast cancer. Recent clinical studies suggest that epigenetic modulation by histone deacetylase (HDAC) inhibitors reverses hormone therapy resistance. However, little is known about epigenetic modulation of the ER during acquired hormone resistance. Our recent phase II study demonstrated that HDAC inhibitors re-sensitize hormone therapy-resistant tumors to the anti-estrogen tamoxifen. In this study, we sought to understand the mechanism behind the efficacy of this combination. Methods We generated cell lines resistant to tamoxifen, named TAMRM and TAMRT, by continuous exposure of ER-positive MCF7 and T47D cells, respectively to 4-hydroxy tamoxifen for over 12 months. HDAC inhibition, along with pharmacological and genetic manipulation of key survival pathways, including ER and Bcl-2, were used to characterize these resistant models. Results The TAMRM cells displayed decreased sensitivity to tamoxifen, fulvestrant and estrogen deprivation. Consistent with previous models, ER expression was retained and the gene harbored no mutations. Compared to parental MCF7 cells, ER expression in TAMRM was elevated, while progesterone receptor (PGR) was lost. Sensitivity of ER to ligands was greatly reduced and classic ER response genes were suppressed. This model conveyed tamoxifen resistance through transcriptional upregulation of Bcl-2 and c-Myc, and downregulation of the cell cycle checkpoint protein p21, manifesting in accelerated growth and reduced cell death. Similar to TAMRM cells, the TAMRT cell line exhibited substantially decreased tamoxifen sensitivity, increased ER and Bcl-2 expression and significantly reduced PGR expression. Treatment with HDAC inhibitors reversed the altered transcriptional events and reestablished the sensitivity of the ER to tamoxifen resulting in substantial Bcl-2 downregulation, growth arrest and apoptosis. Selective inhibition of Bcl-2 mirrored these effects in presence of an HDAC inhibitor. Conclusions Our model implicates elevated ER and Bcl-2 as key drivers of anti-estrogen resistance, which can be reversed by epigenetic modulation through HDAC inhibition. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0533-z) contains supplementary material, which is available to authorized users.