Despite the rapidly identified numbers of lncRNA in humans, exploration of the molecular mechanisms of lncRNA is lagging, because the molecular mechanisms of lncRNA can be various and complex in different conditions. In this study, we found a new molecular mechanism for a versatile molecule, MIR22HG. MIR22HG is an lncRNA that contributes to the initiation and progression of many human cancers, including hepatocellular carcinoma (HCC). We report that MIR22HG was downregulated in 120 HCC samples compared with adjacent nontumor liver tissues. More interestingly, decreased expression of MIR22HG in HCC could predict poor prognosis of HCC patients. Knockdown of MIR22HG promoted the growth, migration and invasion of HCC cells. In exploring the molecular mechanism of MIR22HG, we found that MIR22HG functioned as a tumor suppressor in hepatocellular carcinomas, in part through serving as a competing endogenous RNA to modulate the miRNA-10a-5p level. Moreover, NCOR2 was verified to act as the downstream target gene of MIR22HG/miR-10a-5p. In addition, the MIR22HG/miRNA-10a-5p/NCOR2 axis inhibited the activation of the Wnt/β-catenin pathway. Together, our results demonstrated that MIR22HG inhibited HCC progression in part through the miR-10a-5p/NCOR2 signaling axis and might act as a new prognostic biomarker for HCC patients.

Despite receiving rituximab-combined chemotherapy, follicular lymphoma (FL) patients often suffer tumor recurrence and understand that the cause of relapse in FL would thus significantly ameliorate the tumor therapeutics. In the present study, we show that TRA-1-60-expressing cells are a unique population in FL, converge to the conventional stem cell marker Oct3/4 and ALDH1-positive population, and resist current B-lymphoma agents. TRA-1-60 expression was observed in scattered lymphoma cells in FL tissues only as well as in resting B-lymphocytes inside germinal centers. Retrospective comparison between recurrent and cognate primary tissues showed that the number of TRA-1-60-positive cells from rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (R-CHOP)-treated FL had increased relative to primary tissue, a finding corroborated by assays on different rituximab-treated FL cell lines, FL-18 and DOHH2, wherein TRA-positive cell numbers increased over 10-fold compared to the untreated sample. Concordantly, scanty TRA-1-60-positive FL-18 cells implanted s.c. into mice evinced potent tumor-initiating capacity in vivo, where tumors were 12-fold larger in volume (P = 0.0021 < 0.005) and 13-fold heavier in weight (P = 0.0015 < 0.005) compared to those xenografted from TRA-negative cells. To explain these results, gene expression profiling and qPCR analysis indicated that TRA-1-60-positive cells defined a distinct population from that of TRA-negative cells, with upregulation of multiple drug transporters and therapeutic resistance genes. Hence, TRA-1-60-expressing cells in FL are considered to be vigorously intractable against conventional therapeutic agents, which may explain its refractory recurrence.

BACKGROUND/AIM: Podocalyxin, a member of the CD34 family of cell surface sialomucins, is overexpressed in human embryonal carcinoma cell lines, as well as in several cancer types, and is associated with poor prognosis. Podocalyxin variants are associated with an increased risk and aggressiveness of prostate cancer. Herein podocalyxin protein expression in prostate cancer was characterized.
MATERIALS AND METHODS: Expression of podocalyxin as well as of TRA-1-60 and TRA-1-81 antigens was assessed immunohistochemically in 84 radical prostatectomy specimens and in adjacent normal tissues.
RESULTS: Podocalyxin expression and H-scores were considerably higher in prostate tumors compared to normal tissues. High TRA-1-60 and TRA-1-81 staining was detected, however, in a much smaller percentage of prostate tumors, while their expression and H-scores were low in normal tissues. Similar trends for all three proteins were observed in prostatic intraepithelial neoplasia.
CONCLUSION: Overexpression of podocalyxin in prostate cancer renders the protein a putative immunohistochemical marker of prostate cancer that may contribute to stratification of patients for optimal treatment.

BACKGROUND: Fusion genes are known to be drivers of many common cancers, so they are potential markers for diagnosis, prognosis or therapy response. The advent of paired-end RNA sequencing enhances our ability to discover fusion genes. While there are available methods, routine analyses of large number of samples are still limited due to high computational demands.
RESULTS: We develop FuSeq, a fast and accurate method to discover fusion genes based on quasi-mapping to quickly map the reads, extract initial candidates from split reads and fusion equivalence classes of mapped reads, and finally apply multiple filters and statistical tests to get the final candidates. We apply FuSeq to four validated datasets: breast cancer, melanoma and glioma datasets, and one spike-in dataset. The results reveal high sensitivity and specificity in all datasets, and compare well against other methods such as FusionMap, TRUP, TopHat-Fusion, SOAPfuse and JAFFA. In terms of computational time, FuSeq is two-fold faster than FusionMap and orders of magnitude faster than the other methods.
CONCLUSIONS: With this advantage of less computational demands, FuSeq makes it practical to investigate fusion genes in large numbers of samples. FuSeq is implemented in C++ and R, and available at https://github.com/nghiavtr/FuSeq for non-commercial uses.

Endogenous retroviruses (ERVs) are ancient viral elements that have accumulated in the genome through retrotransposition events. Although they have lost their ability to transpose, many of the long terminal repeats (LTRs) that originally flanked full-length ERVs maintain the ability to regulate transcription. While these elements are typically repressed in somatic cells, they can function as transcriptional enhancers and promoters when this repression is lost. Epstein-Barr virus (EBV), which transforms primary B cells into continuously proliferating cells, is a tumor virus associated with lymphomas. We report here that transformation of primary B cells by EBV leads to genome-wide activation of LTR enhancers and promoters. The activation of LTRs coincides with local DNA hypomethylation and binding by transcription factors such as RUNX3, EBF1, and EBNA2. The set of activated LTRs is unique to transformed B cells compared with other cell lines known to have activated LTRs. Furthermore, we found that LTR activation impacts the B cell transcriptome by up-regulating transcripts driven by cryptic LTR promoters. These transcripts include genes important to oncogenesis of Hodgkin lymphoma and other cancers, such as

The bromodomain and extra-terminal domain (BET) protein BRD4 is emerging as a promising anticancer therapeutic target. However, resistance to BET inhibitors often occurs, and it has been linked to aberrant degradation of BRD4 protein in cancer. Here, we demonstrate that the deubiquitinase DUB3 binds to BRD4 and promotes its deubiquitination and stabilization. Expression of DUB3 is transcriptionally repressed by the NCOR2-HDAC10 complex. The NCOR2 gene is frequently deleted in castration-resistant prostate cancer patient specimens, and loss of NCOR2 induces elevation of DUB3 and BRD4 proteins in cancer cells. DUB3-proficient prostate cancer cells are resistant to the BET inhibitor JQ1 in vitro and in mice, but this effect is diminished by DUB3 inhibitory agents such as CDK4/6 inhibitor in a RB-independent manner. Our findings identify a previously unrecognized mechanism causing BRD4 upregulation and drug resistance, suggesting that DUB3 is a viable therapeutic target to overcome BET inhibitor resistance in cancer.

Lung adenocarcinoma is a major form of lung cancer, which is the leading cause of cancer death. Histone methylation reader proteins mediate the effect of histone methylation, a hallmark of epigenetic and transcriptional regulation of gene expression. However, their roles in lung adenocarcinoma are poorly understood. Here, our bioinformatic screening and analysis in search of a lung adenocarcinoma-promoting histone methylation reader protein show that heterochromatin protein 1γ (HP1γ; also called CBX3) is among the most frequently overexpressed and amplified histone reader proteins in human lung adenocarcinoma, and that high

The development of genomic editing technologies expands the landscape of T cell engineering for adoptive cell therapy. Among the multiple tools that can be used, CRISPR/Cas9 has been shown to be relatively easy to use, simple to design and cost effective with highly efficient multiplex genome engineering capabilities. Allogeneic universal chimeric antigen receptor (CAR) T cells can be produced by disrupting T cell receptor (TCR) and beta-2-microglobulin (B2M) in CAR T cells or by directly knocking in a CAR at the disrupted TRAC locus. The anti-tumor function can be further boosted by simultaneous ablation of PD-1 and CTLA-4. The anti-tumor activities and safety of TCR-transferred T cells can be improved by knocking out endogenous TCR, which avoids the use of affinity-enhanced TCRs that may lose specificity and cause severe adverse effects. Therefore, CRISPR/Cas9 technology holds enormous promise to advance the field of adoptive cell therapy.

Gene editing can be used to overcome allo-recognition, which otherwise limits allogeneic T cell therapies. Initial proof-of-concept applications have included generation of such "universal" T cells expressing chimeric antigen receptors (CARs) against CD19 target antigens combined with transient expression of DNA-targeting nucleases to disrupt the T cell receptor alpha constant chain (TRAC). Although relatively efficient, transgene expression and editing effects were unlinked, yields variable, and resulting T cell populations heterogeneous, complicating dosing strategies. We describe a self-inactivating lentiviral "terminal" vector platform coupling CAR expression with CRISPR/Cas9 effects through incorporation of an sgRNA element into the ΔU3 3' long terminal repeat (LTR). Following reverse transcription and duplication of the hybrid ΔU3-sgRNA, delivery of Cas9 mRNA resulted in targeted TRAC locus cleavage and allowed the enrichment of highly homogeneous (>96%) CAR

T cell malignancies represent a group of hematologic cancers with high rates of relapse and mortality in patients for whom no effective targeted therapies exist. The shared expression of target antigens between chimeric antigen receptor (CAR) T cells and malignant T cells has limited the development of CAR-T because of unintended CAR-T fratricide and an inability to harvest sufficient autologous T cells. Here, we describe a fratricide-resistant "off-the-shelf" CAR-T (or UCART7) that targets CD7+ T cell malignancies and, through CRISPR/Cas9 gene editing, lacks both CD7 and T cell receptor alpha chain (TRAC) expression. UCART7 demonstrates efficacy against human T cell acute lymphoblastic leukemia (T-ALL) cell lines and primary T-ALL in vitro and in vivo without the induction of xenogeneic GvHD. Fratricide-resistant, allo-tolerant "off-the-shelf" CAR-T represents a strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T cell lymphoma without a requirement for autologous T cells.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer worldwide. The pregnane X receptor (PXR) is a nuclear receptor regulating several target genes associated with cancer malignancy. We here demonstrated a significant effect of PXR on HNSCC cell growth, as evidenced in PXR knock-down experiments. PXR transcriptional activity is more importantly regulated by the presence of coactivators and corepressors than by PXR protein expression. To date, there is scarce information on the regulation of PXR in HNSCC and on its role in the pathogenesis of this disease. Coactivator and corepressor expression was screened through qRT-PCR in 8 HNSCC cell lines and correlated to PXR activity, determined by using a reporter gene assay. All cell lines considerably expressed all the cofactors assessed. PXR activity negatively correlated with nuclear receptor corepressor 2 (NCoR2) expression, indicating a major role of this corepressor in PXR modulation and suggesting its potential as a surrogate for PXR activity in HNSCC. To test the association of NCoR2 with the malignant phenotype, a subset of three cell lines was transfected with an over-expression plasmid for this corepressor. Subsequently, cell growth and chemoresistance assays were performed. To elucidate the mechanisms underlying NCoR2 effects on cell growth, caspase 3/7 activity and protein levels of cleaved caspase 3 and PARP were evaluated. In HNO97 cells, NCoR2 over-expression decreased cell growth, chemoresistance and increased cleaved caspase 3 levels, caspase activity and cleaved PARP levels. On the contrary, in HNO124 and HNO210 cells, NCoR2 over-expression increased cell growth, drug resistance and decreased cleaved caspase 3 levels, caspase activity and cleaved PARP levels. In conclusion, we demonstrated a role of PXR and NCoR2 in the modulation of cell growth in HNSCC. This may contribute to a better understanding of the highly variable HNSCC therapeutic response.

T-cell acute lymphoblastic leukemia (T-ALL) is a clonal malignancy of immature T cells. Recently, the next-generation sequencing approach has allowed systematic identification of molecular features in pediatric T-ALL. Here, by performing RNA-sequencing and other genomewide analysis, we investigated the genomic landscape in 61 adult and 69 pediatric T-ALL cases. Thirty-six distinct gene fusion transcripts were identified, with

PURPOSE: In this study, we aimed to assess the association between MYBL2 expression/transcription regulatory activity (TRA) and overall survival (OS) in patients with primary hepatocellular carcinoma (HCC) and to explore the factors related to B-Myb TRA.
MATERIALS AND METHODS: Bioinformatic analysis was performed based on data from the cancer genome atlas-liver hepatocellular carcinoma (TCGA-LIHC) and the human protein atlas (HPA).
RESULTS: The death group in TCGA-LIHC had significantly higher MYBL2 RNA and exon expression than the censor group. The high MYBL2 RNA and exon expression groups had significantly worse OS (P<0.01). Univariate and multivariate analysis confirmed that high MYBL2 expression was an independent prognostic factor of unfavourable OS (HR=1.591, 95%CI: 1.119-2.262, P=0.01). One hundred and fourteen out of 188 primary HCC cases in TCGA-LIHC had elevated transcription of B-Myb's downstream genes. High B-Myb TRA was associated with poor OS (P=0.013). Elevated expression of MYBL2, LIN9, LIN52 and FOXM1 were related to the higher TRA of B-Myb in HCC.
CONCLUSION: High MYBL2 expression/TRA are associated with inferior OS in patients with primary HCC. Increased expression of MYBL2, LIN9, LIN52 and FOXM1 are related to higher TRA of B-Myb in HCC.

The molecular biology of primary nodal T- and NK-cell lymphoma and its relationship with extranodal NK/T-cell lymphoma, nasal type is poorly understood. In this study, we assessed the relationship between nodal and extranodal Epstein-Barr virus-positive T/NK-cell lymphomas using gene expression profiling and copy number aberration analyses. We performed gene expression profiling and copy number aberration analysis on 66 cases of Epstein-Barr virus-associated T/NK-cell lymphoma from nodal and extranodal sites, and correlated the molecular signatures with clinicopathological features. Three distinct molecular clusters were identified with one enriched for nodal presentation and loss of 14q11.2 (TCRA loci). T/NK-cell lymphomas with a nodal presentation (nodal-group) were significantly associated with older age, lack of nasal involvement, and T-cell lineage compared to those with an extranodal presentation (extranodal-group). On multivariate analysis, nodal presentation was an independent factor associated with short survival. Comparing the molecular signatures of the nodal and extranodal groups it was seen that the former was characterized by upregulation of PD-L1 and T-cell-related genes, including CD2 and CD8, and downregulation of CD56, consistent with the CD8

Loss of function mutations in the neurofibromatosis Type 2 (NF2) gene, coding for a tumour suppressor, Merlin, cause multiple tumours of the nervous system such as schwannomas, meningiomas and ependymomas. These tumours may occur sporadically or as part of the hereditary condition neurofibromatosis Type 2 (NF2). Current treatment is confined to (radio) surgery and no targeted drug therapies exist. NF2 mutations and/or Merlin inactivation are also seen in other cancers including some mesothelioma, breast cancer, colorectal carcinoma, melanoma and glioblastoma. To study the relationship between Merlin deficiency and tumourigenesis, we have developed an in vitro model comprising human primary schwannoma cells, the most common Merlin-deficient tumour and the hallmark for NF2. Using this model, we show increased expression of cellular prion protein (PrP

BACKGROUND: Rhazya stricta Decne. is a medicinal plant that is widespread in Saudi Arabia and desert areas of the Arabian Peninsula. Its extract contains alkaloids, tannins, and flavonoids that are involved in different biological activities. The study aim was to evaluate the effects of Rhazya stricta plant extracts on the proliferation and differentiation of NTERA-2 (NT2) pluripotent embryonal carcinoma cells.
METHODS: Soxhlet extraction was carried out using different solvents to extract stems, leaves and fruit parts of this plant. Cytotoxicity was evaluated by an MTS cell viability assay. The ability of the plant extract to induce cell differentiation was examined phenotypically using an inverted light microscope. The expression of pluripotency markers was investigated by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. Phytochemical screening of chloroform stem extracts was carried out and a chromatographic fingerprint was generated using gas chromatography - mass spectrometry (GC-MS).
RESULTS: Chloroform stem extract induced differentiation of NT2 cells at 5 μg/ml, and the differentiated cells exhibited neurite formation. Following induction of differentiation, there was significant down-regulation of the pluripotency marker genes Oct4 and Sox2. In addition, the surface antigen pluripotency marker, TRA-1-60, was strongly down-regulated. Phytochemical analysis of the extract showed the presence of alkaloids and saponins. The chromatogram revealed the presence of fifteen compounds with different retention times.
CONCLUSION: Our results demonstrate for the first time that chloroform stem extract of R. stricta can induce neuronal differentiation of stem cells at an early stage and may contain potential therapeutic agent that can be used in neurodegenerative diseases.

The molecular mechanisms underlying the opposing functions of glucocorticoid receptors (GRs) and estrogen receptor α (ERα) in breast cancer development remain poorly understood. Here we report that, in breast cancer cells, liganded GR represses a large ERα-activated transcriptional program by binding, in trans, to ERα-occupied enhancers. This abolishes effective activation of these enhancers and their cognate target genes, and it leads to the inhibition of ERα-dependent binding of components of the MegaTrans complex. Consistent with the effects of SUMOylation on other classes of nuclear receptors, dexamethasone (Dex)-induced trans-repression of the estrogen E

OBJECTIVE: The present study aimed to establish an induced pluripotent stem cell (iPSC) line from acute myelogenous leukemia (AML) cells in vitro and identify their biological characteristics.
METHODS: Cells from the AML-infiltrated skin from an M6 patient were infected with a lentivirus carrying OCT4, SOX2, KLF4 and C-MYC to induce iPSCs. The characteristics of the iPSCs were confirmed by alkaline phosphatase (ALP) staining. The proliferation ability of iPSCs was detected with a CCK-8 assay. The expression of pluripotency markers was measured by immunostaining, and the expression of stem cell-related genes was detected by qRT-PCR; distortion during the induction process was detected by karyotype analysis; the differentiation potential of iPSCs was determined by embryoid body-formation and teratoma-formation assays. ALP staining confirmed that these cells exhibited positive staining and had the characteristics of iPSCs.
RESULTS: The CCK-8 assay showed that the iPSCs had the ability to proliferate. Immunostaining demonstrated that iPSC clones showed positive expression of NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. qRT-PCR results revealed that the mRNA expression of Nanog, Lin28, Cripto, FOX3, DNMT3b, DPPA2, and DPPA4 significantly increased in iPSCs. Karyotype analysis found no chromosome aberration in the iPSCs. The results of the embryoid body-formation and teratoma-formation assays indicated that the iPSCs had the potential to differentiate into all three germ layers.
CONCLUSION: Our study provided evidence that an iPSC line derived from AML cells was successfully established.

BACKGROUND: Patients with germline mutations in a tumour suppressor gene called CYLD develop multiple, disfiguring, hair follicle tumours on the head and neck. The prognosis is poor, with up to one in four mutation carriers requiring complete surgical removal of the scalp. There are no effective medical alternatives to treat this condition. Whole genome molecular profiling experiments led to the discovery of an attractive molecular target in these skin tumour cells, named tropomyosin receptor kinase (TRK), upon which these cells demonstrate an oncogenic dependency in preclinical studies. Recently, the development of an ointment containing a TRK inhibitor (pegcantratinib - previously CT327 - from Creabilis SA) allowed for the assessment of TRK inhibition in tumours from patients with inherited CYLD mutations.
METHODS/DESIGN: Tropomysin Receptor Antagonism in Cylindromatosis (TRAC) is a two-part, exploratory, early phase, single-centre trial. Cohort 1 is a phase 1b open-labelled trial, and cohort 2 is a phase 2a randomised double-blinded exploratory placebo-controlled trial. Cohort 1 will determine the safety and acceptability of applying pegcantratinib for 4 weeks to a single tumour on a CYLD mutation carrier that is scheduled for a routine lesion excision (n = 8 patients). Cohort 2 will investigate if CYLD defective tumours respond following 12 weeks of treatment with pegcantratinib. As patients have multiple tumours, we intend to treat 10 tumours in each patient, 5 with active treatment and 5 with placebo. Patients will be allocated both active and placebo treatments to be applied randomly to tumours on the left or right side. The target is to treat 150 tumours in a maximum of 20 patients. Tumour volume will be measured at baseline and at 4 and 12 weeks. The primary outcome measure is the proportion of tumours responding to treatment by 12 weeks, based on change in tumour volume, with secondary measures based on adverse event profile, treatment compliance and acceptability, changes in tumour volume and surface area, patient quality of life and pain.
DISCUSSION: Interventions for rare genetic skin diseases are often difficult to assess in an unbiased way due to small patient numbers and the challenges of incorporating adequate controls into trial design. Here we present a single-centre, randomised, placebo-controlled trial design that leverages the multiplicity of tumours seen in an inherited skin tumour syndrome that may inform the design of other studies in similar genetic diseases.
TRIAL REGISTRATION: International Standard Randomised Controlled Trial Number Registry, ISRCTN75715723 . Registered on 22 October 2014.

Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumour rejection. The most successful CARs used to date are those targeting CD19 (ref. 2), which offer the prospect of complete remission in patients with chemorefractory or relapsed B-cell malignancies. CARs are typically transduced into the T cells of a patient using γ-retroviral vectors or other randomly integrating vectors, which may result in clonal expansion, oncogenic transformation, variegated transgene expression and transcriptional silencing. Recent advances in genome editing enable efficient sequence-specific interventions in human cells, including targeted gene delivery to the CCR5 and AAVS1 loci. Here we show that directing a CD19-specific CAR to the T-cell receptor α constant (TRAC) locus not only results in uniform CAR expression in human peripheral blood T cells, but also enhances T-cell potency, with edited cells vastly outperforming conventionally generated CAR T cells in a mouse model of acute lymphoblastic leukaemia. We further demonstrate that targeting the CAR to the TRAC locus averts tonic CAR signalling and establishes effective internalization and re-expression of the CAR following single or repeated exposure to antigen, delaying effector T-cell differentiation and exhaustion. These findings uncover facets of CAR immunobiology and underscore the potential of CRISPR/Cas9 genome editing to advance immunotherapies.

Tumor-infiltrating CD8

Human iPSC line TSD-01-hiPSC was generated from fibroblasts of a patient with infantile Tay-Sachs disease (TSD). The patient is compound heterozygous at the HEXA gene by carrying a 1278insTATC allele and an IVS12+1G>C allele. STEMCCA lentivirus, which expresses OCT4, SOX2, KLF4, and c-MYC from a polycistronic transcript, were used for reprogramming. TSD-01-hiPSC express pluripotency markers such as OCT4, SOX2, NANOG, Tra-1-60, and alkaline phosphatase, and can differentiate into tissues from all the three embryonic germ layers. This TSD patient-derived hiPSC line may serve as a valuable in vitro tool for disease modeling and drug test.

Retinoblastoma is the most common intraocular malignancy in pediatric patients. It develops rapidly in the retina and can be fatal if not treated promptly. It has been proposed that a small population of cancer cells, termed cancer stem cells (CSCs), initiate tumorigenesis from immature tissue stem cells or progenitor cells. Reprogramming technology, which can convert mature cells into pluripotent stem cells (iPS), provides the possibility of transducing malignant cancer cells back to CSCs, a type of early stage of cancer. We herein took advantage of reprogramming technology to induce CSCs from retinoblastoma cancer cells. In the present study, the 4 Yamanaka transcription factors, Oct4, Sox2, Klf4 and c-myc, were transduced into retinoblastoma cells (Rbc51). iPS-like colonies were observed 15 days after transduction and showed significantly enhanced CSC properties. The gene and protein expression levels of pluripotent stem cell markers (Tra-1-60, Oct4, Nanog) and cancer stem cell markers (CD133, CD44) were up-regulated in transduced Rbc51 cells compared to control cells. Moreover, iPS-like CSCs could be sorted using the Magnetic-activated cell sorting (MACS) method. A sphere formation assay demonstrated spheroid formation in transduced Rbc51 cells cultured in serum free media, and these spheroids could be differentiated into Pax6-, Nestin-positive neural progenitors and rhodopsin- and recoverin-positive mature retinal cells. The cell viability after 5-Fu exposure was higher in transduced Rbc51 cells. In conclusion, CSCs were generated from retinoblastoma cancer cells using reprogramming technology. Our novel method can generate CSCs, the study of which can lead to better understanding of cancer-specific initiation, cancer epigenetics, and the overlapping mechanisms of cancer development and pluripotent stem cell behavior.

Somatic mutations in CREBBP occur frequently in B-cell lymphoma. Here, we show that loss of CREBBP facilitates the development of germinal center (GC)-derived lymphomas in mice. In both human and murine lymphomas, CREBBP loss-of-function resulted in focal depletion of enhancer H3K27 acetylation and aberrant transcriptional silencing of genes that regulate B-cell signaling and immune responses, including class II MHC. Mechanistically, CREBBP-regulated enhancers are counter-regulated by the BCL6 transcriptional repressor in a complex with SMRT and HDAC3, which we found to bind extensively to MHC class II loci. HDAC3 loss-of-function rescued repression of these enhancers and corresponding genes, including MHC class II, and more profoundly suppressed CREBBP-mutant lymphomas in vitro and in vivo Hence, CREBBP loss-of-function contributes to lymphomagenesis by enabling unopposed suppression of enhancers by BCL6/SMRT/HDAC3 complexes, suggesting HDAC3-targeted therapy as a precision approach for CREBBP-mutant lymphomas.
SIGNIFICANCE: Our findings establish the tumor suppressor function of CREBBP in GC lymphomas in which CREBBP mutations disable acetylation and result in unopposed deacetylation by BCL6/SMRT/HDAC3 complexes at enhancers of B-cell signaling and immune response genes. Hence, inhibition of HDAC3 can restore the enhancer histone acetylation and may serve as a targeted therapy for CREBBP-mutant lymphomas. Cancer Discov; 7(1); 38-53. ©2016 AACR.See related commentary by Höpken, p. 14This article is highlighted in the In This Issue feature, p. 1.

Approximately 70% of breast cancers express the estrogen receptor (ER)α and are treated with the ERα antagonist, tamoxifen. However, resistance to tamoxifen frequently develops in advanced breast cancer, in part due to a down-regulation of ERα corepressors. Nuclear receptor corepressors function by attenuating hormone responses and have been shown to potentiate tamoxifen action in various biological systems. Recent genomic data on breast cancers has revealed that genetic and/or genomic events target ERα corepressors in the majority of breast tumors, suggesting that the loss of nuclear receptor corepressor activity may represent an important mechanism that contributes to intrinsic and acquired tamoxifen resistance. Here, the biological functions of ERα corepressors are critically reviewed to elucidate their role in modifying endocrine sensitivity in breast cancer. We highlight a mechanism of gene repression common to corepressors previously shown to enhance the antitumorigenic effects of tamoxifen, which involves the recruitment of histone deacetylases (HDACs) to DNA. As an indicator of epigenetic disequilibrium, the loss of ERα corepressors may predispose cancer cells to the cytotoxic effects of HDAC inhibitors, a class of drug that has been shown to effectively reverse tamoxifen resistance in numerous studies. HDAC inhibition thus appears as a promising therapeutic approach that deserves to be further explored as an avenue to restore drug sensitivity in corepressor-deficient and tamoxifen-resistant breast cancers.

Adoptive transfer of T cells with engineered T-cell receptor (TCR) genes that target tumor-specific antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. To test this hypothesis, we developed a multistep strategy that involved TCRB deep sequencing of the CD8(+)PD-1(+) T-cell subset, matching of TCRA-TCRB pairs by pairSEQ and single-cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes, which included reactivity against neoantigens. These data show the feasibility of developing a rapid, personalized TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity. Cancer Immunol Res; 4(9); 734-43. ©2016 AACR.

Current evidence suggests that the majority of DCIS lesions do not progress to invasive carcinoma, and overtreatment of DCIS is a significant problem. We previously reported an 8-gene signature that differentiated microdissected low-grade (LG) DCIS lesions with and without associated stromal invasion, based on differential DNA copy number changes detected by quantitative (q) PCR. The current study was undertaken to validate our candidate breast cancer invasion gene panel in a larger series of non-microdissected LG DCIS cases, and to investigate its potential utility in intermediate-grade (IG) and high-grade (HG) DCIS. Representative paraffin blocks were selected from 267 resected DCIS cases with 5-15 years of follow-up (139 pure DCIS ["PD"] and 128 mixed DCIS with associated invasion ["MD"]). These included 171 LG, 46 IG and 50 HG DCIS cases. Gene copy number changes were determined by qPCR, and their differential distribution in the PD and MD subgroups was evaluated. As an alternate platform, we employed immunohistochemistry (IHC). Novel IHC assays were developed for all eight candidate genes, and increased or reduced protein expression was manually scored. Separate multi-gene models were developed for qPCR and IHC to distinguish progressing and non-progressing DCIS lesions. By qPCR analysis, a panel of six genes, as well as CELSR1 alone (a potential invasion suppressor), differentiated PD and MD cases in LG and IG, but not in HG DCIS. By IHC, a panel of three genes, as well as GRAP2 alone (a potential invasion promoter), also distinguished PD and MD cases in LG and IG, but not in HG DCIS. The combination of CELSR1 (by qPCR) and GRAP2 (by IHC) had the best discriminatory power (p = 0.00004). Assays testing either or both of these genes have the potential to become important adjuncts for choosing appropriate treatment for LG/IG DCIS patients.

Long noncoding RNAs (lncRNAs) dysregulated in cancer potentially play oncogenic or tumor-suppressive roles. While the X inactivate-specific transcript (Xist) lncRNA is important for X-chromosome inactivation in female cells, very little is known about the role of Xist in human breast cancer in modulating cellular pathway(s). Here, we show that Xist expression is significantly reduced in breast tumor samples and cancer cell lines. Xist knockdown or overexpression resulted in increased or decreased levels, respectively, of AKT phosphorylation and cell viability. Further studies revealed an inverse correlation between Xist and phospho-AKT levels in breast cancer samples. Additionally, Xist knockdown-elicited increase of cell viability was attenuated by AKT inhibitor. These results suggest that Xist negatively regulates cell viability via inhibition of AKT activation. Interestingly, decreased Xist expression in breast cancer samples was associated with reduced levels of Jpx RNA, an lncRNA that positively regulates Xist promoter activity. Accordingly, Jpx knockdown enhanced AKT activation and cell viability. We also demonstrate that knockdown of Xist or SPEN, an intermediator protein to link Xist, SMRT co-repressor and HDAC3 complexes for X-chromosome inactivation, decreased expression of PHLPP1, a phosphatase to remove AKT phosphorylation, via increased HDAC3 recruitment to the PHLPP1 promoter, correlating with increased AKT phosphorylation. Our findings elucidate the tumor suppressor role of Xist in breast cancer and provide the molecular basis of Xist in downregulating AKT activation.

OBJECTIVE: Congenital generalized lipodystrophy (CGL) is an autosomal recessive disorder characterized by marked scarcity of adipose tissue, extreme insulin resistance, hypertriglyceridemia, hepatic steatosis and early-onset diabetes. Mutation of the BSCL2/SEIPIN gene causes the most severe form of CGL. The aim of this study was to generate induced pluripotent stem (iPS) cells from patients with CGL harboring BSCL2/SEIPIN mutations.
METHODS: Skin biopsies were obtained from two Japanese patients with CGL harboring different nonsense mutations (E189X and R275X) in BSCL2/SEIPIN. The fibroblasts thus obtained were infected with retroviruses encoding OCT4, SOX2, c-MYC, and KLF4. The generated iPS cells were evaluated for pluripotency by examining the expression of pluripotency markers (alkaline phosphatase, SSEA-4, TRA-1-60, and NANOG) and their ability to differentiate to three germ layers in vitro by forming embryoid bodies, and to form teratomas in vivo. Adipogenic capacity of differentiated BSCL2-iPS cells was determined by oil red O and adipose differentiation-related protein (ADRP) staining. Rescue experiments were also performed using stable expression of wild-type BSCL2. A coimmunoprecipitation assay was conducted to investigate the interaction of SEIPIN with ADRP.
RESULTS: iPS cells were generated from fibroblasts of the two patients with CGL. Each of the patient-derived iPS (BSCL2-iPS) clones showed all of the hallmarks of pluripotency and could differentiate into derivatives of all three germ layers in vitro by forming embryoid bodies, and form teratomas after injection into mouse testes. BSCL2-iPS cells maintained the mutations in BSCL2 and lacked intact BSCL2. Upon adipogenic differentiation, BSCL2-iPS cells exhibited marked reduction of lipid droplet formation concomitant with diffuse cytoplasmic distribution of ADRP, compared with iPS cells from healthy individuals. Forced expression of BSCL2 not only rescued the lipid accumulation defects, but also restored cytoplasmic punctate localization of ADRP in BSCL2-iPS cells. Coimmunoprecipitation indicated SEIPIN interacted with ADRP.
CONCLUSION: BSCL2-iPS cells that recapitulate the lipodystrophic phenotypes in vitro could provide valuable models with which to study the physiology of lipid accumulation and the pathology of human lipodystrophy. We found that BSCL2 defines the localization of ADRP, which has a role in lipid accumulation and adipogenic differentiation.

BACKGROUND: Abdominoperineal excision is performed in patients with locally advanced, low rectal carcinoma. Reconstruction of the dorsal vagina and perineum using the vertical rectus abdominis myocutaneous flap following extensive surgery results in favorable surgical outcome and quality of life. However, the rectus abdominis muscle, as part of the anterior abdominal wall, may develop fibrous lesions also as a transplant.
CASE PRESENTATION: A 39-year-old female patient with low rectal cancer and extensive colorectal polyposis was treated with neoadjuvant chemoradiotherapy followed by colectomy and abdominoperineal excision with resection of the dorsal vaginal wall and subsequent reconstruction of the perineum using the vertical rectus abdominis myocutaneous flap. At the 6-month follow-up, a suspected 2 × 2 cm tumor recurrence was detected in the transposed tissue and was subsequently surgically removed. Histologic examination concluded with fibromatosis. Genetic testing revealed a known disease-causing mutation in the adenomatous polyposis coli gene, confirming the diagnosis of familial adenomatous polyposis.
CONCLUSIONS: Fibromatosis may affect the anterior abdominal wall, that is the rectus abdominis muscle, at the primary site or may develop in the muscle after its transposition into the perineum at pelvic reconstruction. Fibromatosis in the muscle flap after pelvic reconstruction may present a difficult diagnostic challenge for the multidisciplinary team.