Search

Vascular smooth muscle cell (SMC) phenotypic modulation contributes to the development of restenosis. A sorafenib-eluting stent was specifically designed to target SMC phenotypic modulation to inhibit in-stent restenosis in the present study. SMC contractile protein from the freshly isolated rat aorta was expressed at a high level, but its expression was dramatically reduced after SMCs were cultured in 10% FBS for 1 wk. After sorafenib treatment, SMC contractile protein expression was markedly upregulated. We further observed that Yes-associated protein (YAP) expression was attenuated after sorafenib treatment in a dose-dependent manner. Overexpression of YAP by lentivirus reversed the expression of sorafenib-induced SMC contractile protein and increased the expression of cyclin D. Mechanistically, sorafenib regulated the serum response factor-myocardin (SRF-Myocd) complex through competitive binding of YAP to Myocd and increased SRF binding to CArG-containing regions of SMC-specific contractile genes within intact chromatin, thereby controlling the activity of smooth muscle-specific gene transcription. In a rabbit carotid model, the sorafenib-eluting stent (SFES) dramatically inhibited in-stent restenosis and upregulated SMC contractile protein expression. Overexpression of YAP blocked the antirestenosis effect of SFES and repressed contractile smooth muscle-specific genes in vivo, indicating that SFES attenuated in-stent restenosis through YAP-mediated SMC phenotypic modulation. We demonstrated that SFES attenuated in-stent restenosis through YAP-mediated SMC phenotypic modulation. Targeting SMC phenotypic modulation by drug-eluting stent represents an attractive therapeutic approach for the treatment of occlusive vascular diseases. NEW & NOTEWORTHY In the present study, we demonstrated that sorafenib regulates smooth muscle cell (SMC) phenotypic modulation from a proliferative to a contractile state. Sorafenib induced a myocardin-serum response factor interaction and increased SMC contractile gene transcription through the Yes-associated protein pathway. Moreover, local delivery of sorafenib regulating SMC phenotypic modulation represents a promising strategy in the design of drug-eluting stents.

Purpose: To report a novel internal and external blood bypass method for cerebral protection during in situ triple-fenestrated stent-graft repair in the aortic arch. Technique: A method was devised to combine internal and external blood bypass circuits to preserve cerebral blood flow when all 3 supra-aortic branches are covered by the stent-graft. Long 14-F to 18-F introducers are placed retrogradely into the right and left common carotid arteries (CCAs). Smaller sheaths are placed antegradely into the internal carotid arteries (ICAs) bilaterally and into the right CCA introducer, which has had an aperture cut into it for flow to pass into the smaller sheath. The right CCA introducer is positioned in the ascending aorta to supply the innominate artery; the smaller sheath in the right CCA introducer is positioned at the aperture. The small sheath in the right CCA is connected to the left ICA sheath, and the left CCA fenestration is made and stented. The left ICA sheath is connected to the right ICA sheath, and the right CCA introducer is pulled back to the origin of the innominate artery and the smaller sheath removed. The fenestrations for the innominate and left subclavian arteries are opened sequentially. In 8 patients, complete bypass to maintain brain perfusion was performed for an average 17.6±6.9 minutes; intraoperative transcranial Doppler monitoring during flow bypass showed no notable decline in intracranial blood flow velocity. Two patients suffered stroke; one recovered completely. Conclusion: A cerebral protection strategy that integrates internal and external blood flow bypass techniques to maintain adequate brain blood flow is simple and feasible for in situ triple-fenestration aortic arch stent-graft repairs. However, neurological complications were not avoided with this method; thus, further research and development are required.

Objectives The aim of this study was to analyze the short-term outcomes of in situ fenestration and discuss its feasibility and safety for the treatment of aortic dissection or aneurysm involving aortic arch. Methods A retrospective single-center review was conducted on patients who were treated with ISF technique to revascularize supra-arch branches from Jun 2017 to Oct 2019. Computed tomographic angiography (CTA) was performed to assess the patency of bridging stents, endoleaks and prognosis prior to discharge, after 3 months, 6 months, 12 months and yearly thereafter. Patient demographics, operative details, clinical outcomes, and complications were analyzed and then discussed in this paper. Results A total of 21 patients were diagnosed with arch pathologies, 5 type A aortic dissections, 12 type B aortic dissections and 4 thoracic aortic aneurysms. There were 19 men and 2 women (mean age 60.7 ± 15.3). 8 cases were treated with t hree-fenestration stent grafts, 1 case with two-fenestration stent graft, and 12 case with single-fenestration stent grafts. Overall technical success rate was 95.2%. Mean operation time was 227.4 ± 143.8 min. Complications were intraoperative hemorrhage (>1000 ml, 2), stroke (2), hydropericardium (1) and endoleaks (2 type Ⅲ, 1 type Ⅰ). There was no aorta-related mortality or late endoleaks during the mean follow-up of 25.5 ± 6.2 months. All the bridging stents remained patent and there was no migration according to follow-up CTA. Conclusion With low complication and mortality rate, ISF is an effective and feasible method for the total endovascular aortic arch repair. Long-term follow-up study is needed to evaluate its durability.

To report the early results of castor device, a kind of unibody single-branched stent graft, in the treatment of type B aortic dissection (TBAD) involving the LSA. From April 2013 to February 2014, 21 patients with TBADs underwent TEVAR with LSA revascularization by unibody single-branched stent grafts. Three patients with penetrating aortic ulcers in the aortic arch received additional reconstruction of left common carotid artery with chimney technique. Follow-up evaluations were conducted with computed tomography angiography (CTA) at 6, 12 months and annually after that. All of the proximal entry tears were completely excluded. Good patency of the grafts was found in all cases. A small type I endoleak occurred in one patient during the procedure. Perioperative mortality is null, and there was no occurrence of serious complications. All patients completed the follow-up except one lost contact after discharge. One death occurred within 6 months after the operation, resulting from myocardial infarction, considered unrelated to the stent implantation. No endoleak occurred during follow-up. One compression of a chimney stent and one twist of side branch graft of castor were observed in 2 different patients, respectively. In other cases, CTA scans showed good patency of both the branched and chimney grafts. Two patients had partial thrombosis of the false lumen during follow-up. In other patients, complete thrombosis in the false lumen in thoracic aorta was revealed. The single-branched stent graft was safe and efficient in the 1-year follow-up. Further studies are required to determine its long-term outcomes.

Basal p53 levels are tightly suppressed under normal conditions. Disrupting this regulation results in elevated p53 levels to induce cell cycle arrest, apoptosis, and tumor suppression. Here, we report the suppression of basal p53 levels by a nuclear, p53-regulated long noncoding RNA that we termed PURPL (p53 upregulated regulator of p53 levels). Targeted depletion of PURPL in colorectal cancer cells results in elevated basal p53 levels and induces growth defects in cell culture and in mouse xenografts. PURPL associates with MYBBP1A, a protein that binds to and stabilizes p53, and inhibits the formation of the p53-MYBBP1A complex. In the absence of PURPL, MYBBP1A interacts with and stabilizes p53. Silencing MYBBP1A significantly rescues basal p53 levels and proliferation in PURPL-deficient cells, suggesting that MYBBP1A mediates the effect of PURPL in regulating p53. These results reveal a p53-PURPL auto-regulatory feedback loop and demonstrate a role for PURPL in maintaining basal p53 levels.

Background Activation of the YAP (Yes‐associated protein) pathway has been demonstrated to be related to smooth muscle cells ( SMC s) phenotypic modulation and vessel restenosis. The aim of this study was to illustrate the molecular mechanisms that regulate the expression of YAP during the process of SMC s phenotypic switch. Whether the molecular basis identified in the study could be a potential therapeutic target for drug‐eluting stents is further tested. Methods and Results In cell culture and in rat carotid arterial injury models, Sp‐1 (specificity protein 1) expression was significantly induced, and correlated with SMC s proliferative phenotype. Overexpression of Sp‐1 promoted SMC s proliferation and migration. Conversely, siSp‐1 transfection or Sp‐1 inhibitor Mithramycin A treatment attenuates SMC proliferation and migration. Through gain‐ and loss‐function assays, we demonstrated that YAP was involved in Sp‐1‐mediated SMC phenotypic switch. Mechanistically, activated Sp‐1 regulated YAP transcriptional expression through binding to its promoter. Moreover, we fabricated a Sp‐1 inhibitor Mithramycin A‐eluting stent and further tested it. In the rabbit carotid model, Mithramycin A‐eluting stent inhibited YAP transcription and attenuated in‐stent restenosis through regulating YAP ‐mediated SMC phenotypic switch. Conclusions Sp‐1 controls phenotypic modulation of SMC by regulating transcription factor YAP . Drug‐eluting stent targeting Sp‐1 might represent a novel therapeutic strategy to prevent in‐stent restenosis.

Agents targeting the insulin-like growth factor-1 receptor (IGF1R) are in clinical development, but, despite some initial success of single agents in sarcoma, response rates are low with brief durations. Thus, it is important to identify markers predictive of response, to understand mechanisms of resistance, and to explore combination therapies. In this study, we found that, although associated with PAX3-FKHR translocation, increased IGF1R level is an independent prognostic marker for worse overall survival, particularly in patients with PAX3-FKHR-positive rhabdomyosarcoma (RMS). IGF1R antibody-resistant RMS cells were generated using an in vivo model. Expression analysis indicated that IGFBP2 is both the most affected gene in the insulin-like growth factor (IGF) signaling pathway and the most significantly downregulated gene in the resistant lines, indicating that there is a strong selection to repress IGFBP2 expression in tumor cells resistant to IGF1R antibody. IGFBP2 is inhibitory to IGF1R phosphorylation and its signaling. Similar to antibodies to IGF1/2 or IGF2, the addition of exogenous IGFBP2 potentiates the activity of IGF1R antibody against the RMS cells, and it reverses the resistance to IGF1R antibody. In contrast to IGF1R, lower expression of IGFBP2 is associated with poorer overall survival, consistent with its inhibitory activity found in this study. Finally, blocking downstream Protein kinase B (AKT) activation with Phosphatidylinositide 3-kinases (PI3K)- or mammalian target of rapamycin (mTOR)-specific inhibitors significantly sensitized the resistant cells to the IGF1R antibody. These findings show that constitutive IGFBP2 downregulation may represent a novel mechanism for acquired resistance to IGF1R therapeutic antibody in vivo and suggest various drug combinations to enhance antibody activity and to overcome resistance.

One-third of all estrogen receptor (ER)–positive breast tumors treated with endocrine therapy fail to respond, and the remainder is likely to relapse in the future. Almost all data on endocrine resistance has been obtained in models of invasive ductal carcinoma (IDC). However, invasive lobular carcinomas (ILC) comprise up to 15% of newly diagnosed invasive breast cancers each year and, whereas the incidence of IDC has remained relatively constant during the last 20 years, the prevalence of ILC continues to increase among postmenopausal women. We report a new model of Tamoxifen (TAM)-resistant invasive lobular breast carcinoma cells that provides novel insights into the molecular mechanisms of endocrine resistance. SUM44 cells express ER and are sensitive to the growth inhibitory effects of antiestrogens. Selection for resistance to 4-hydroxytamoxifen led to the development of the SUM44/LCCTam cell line, which exhibits decreased expression of ERα and increased expression of the estrogen-related receptor γ (ERRγ). Knockdown of ERRγ in SUM44/LCCTam cells by siRNA restores TAM sensitivity, and overexpression of ERRγ blocks the growth-inhibitory effects of TAM in SUM44 and MDA-MB-134 VI lobular breast cancer cells. ERRγ-driven transcription is also increased in SUM44/LCCTam, and inhibition of activator protein 1 (AP1) can restore or enhance TAM sensitivity. These data support a role for ERRγ/AP1 signaling in the development of TAM resistance and suggest that expression of ERRγ may be a marker of poor TAM response. [Cancer Res 2008;68(21):8908–17]

Background: Multiple Endocrine Neoplasia (MEN) 2B is a rare hereditary disorder characterized by medullary thyroid cancer (MTC) in early childhood, pheochromocytoma, and mucosal neuromas. Patients with advanced MTC are treated with rearranged during transfection (RET) targeting tyrosine kinase inhibitors (TKIs) such as vandetanib. Despite initial responses, many patients progress on TKI therapy and mechanisms of resistance are yet to be elucidated. We analyzed tumor samples from seven children with MEN2B and MTC enrolled in a natural history study (NCT01660984). Methods: DNA samples from tumor and adjacent normal tissue from paraffin embedded blocks or unstained slides were analyzed by a custom capture next-generation sequencing panel. Bioinformatics analyses identified point mutations, insertions, deletions and copy number variations. Results: Seven patients (median age at study enrollment 14 years, range 11-17 years) with the RET p.Met918Thr germline mutation were included in analysis. Tumor samples were available for three patients pre-TKI (four samples), two patients at progression on TKI (three samples), and two patients both, pre-TKI and at progression (five samples). Pre-TKI samples exhibited few tumor specific mutations or copy number variations and 4/5 patients had loss of chromosome 1p. Progression for one patient was associated with acquisition of a previously unidentified p.Leu790Phe mutation within the kinase domain of the RET gene. Loss of heterozygosity and increase in copy number variations were noted in 4/5 samples at tumor progression. Two patients had copy number loss of chromosome 14 and three had copy number gain of chromosome 1q. Recurrent, somatic, non-synonymous mutations were not identified in the sample set. Conclusion: In children with MEN2B and MTC, we identified increase in copy number variations and a somatic mutation within the RET gene as potential mechanisms of drug resistance. Our data imply that a common genetic mechanism for progression on TKI may not exist within this small sample set and highlight the need for serial collection of tumor tissue. Further, whole genome aneuploidy may provide rationale for the evaluation of cytotoxic chemotherapy in patients who experience progressive disease on TKI therapy. Analysis of additional samples and whole exome DNA and RNA sequencing are ongoing. Citation Format: Ira L. Kraft, Srivandana Akshintala, Keith J. Killian, Robert B. Hufnagel, John W. Glod, Claudia Derse-Anthony, Yuelin Zhu, Holly S. Stevenson, Diana Bradford, Maria J. Merino, Frank M. Balis, Elizabeth Fox, Brigitte C. Widemann, Jack F. Shern, Paul S. Meltzer. Genomic mechanisms of disease progression in pediatric medullary thyroid cancer (MTC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4882. doi:10.1158/1538-7445.AM2017-4882

Genetic alterations and etiology of thymic epithelial tumors (TETs) are largely unknown, hampering the development of effective targeted therapies for patients with TETs. Here TETs of advanced-stage patients enrolled in a clinical trial of molecularly-guided targeted therapies were employed for targeted sequencing of 197 cancer-associated genes. Comparative sequence analysis of 78 TET/blood paired samples obtained from 47 thymic carcinoma (TC) and 31 thymoma patients revealed a total of 86 somatic non-synonymous sequence variations across 39 different genes in 33 (42%) TETs. TCs (62%; 29/47) showed higher incidence of somatic non-synonymous mutations than thymomas (13%; 4/31; p < 0.0001). TP53 was the most frequently mutated gene in TETs (n = 13; 17%), especially in TCs (26%), and was associated with a poorer overall survival (p < 0.0001). Genes in histone modification [BAP1 (n = 6; 13%), SETD2 (n = 5; 11%), ASXL1 (n = 2; 4%)], chromatin remodeling [SMARCA4 (n = 2; 4%)], and DNA methylation [DNMT3A (n = 3; 7%), TET2 (n = 2; 4%), WT1 (n = 2; 4%)] pathways were recurrently mutated in TCs, but not in thymomas. Our results suggest a potential disruption of epigenetic homeostasis in TCs, and a substantial difference in genetic makeup between TCs and thymomas. Further investigation is warranted into the roles of epigenetic dysregulation in TC development and its potential for targeted therapy.

By means of LSAB and ABC techniques, the expressions of p53, c-erbB-2, EGFR and ras were examined on 75 cases of gastric cancer (GC) specimens.(1) The positive rate of p53, c-erbB-2, EGFR and ras expression was 41.3%, 18.7%, 61.3% and 46.7%, respectively. The early cases and the intestinal type cases of GC had higher p53 positive rates than those of the advanced and the diffuse type ones, respectively. Two of 4 cases of severe dysplasia lesions adjacent to carcinoma sites were positive. c-erbB-2 positive immunoreactivity was only localized in the cancer cells, and tissues adjacent to the carcinoma sites were negative. Higher frequencies of c-erbB-2 expression were found in the well-differentiated GC. Positive correlation was found between EGFR expression and degree of malignancy of GC. (2) No correlation was found between the expression of c-erbB-2 and EGFR. (3) The expression of ras correlated positively with EGFR expression, but negatively with c-erbB-2 (P0.05). (4) No correlation was found between the expression of p53 and other genes examined. The results demonstrate that many gene abnormalities are present in the process of gastrocarcinogenesis.

Many aspects of development, tumor growth and metastasis depend upon the provision of an adequate vasculature. This can be a result of regulated angiogenesis, recruitment of circulating endothelial progenitors and/or vascular trans-differentiation. The present study demonstrates that treatment of SKBR-3 breast cancer cells with retinoic acid (RA), an important regulator of embryogenesis, cancer and other diseases, stimulates the formation of networks in Matrigel. RA-treatment of SKBR-3 cells co-cultured with human umbilical vein endothelial cells resulted in the formation of mixed structures. RA induces expression of many endothelial genes including vascular endothelial (VE) cadherin. VE-cadherin was also induced by RA in a number of other breast cancer cells. We show that RA-induced VE-cadherin is responsible for the RA-induced morphological changes. RA rapidly induced the expression of Sox-9 and ER81, which in turn form a complex on the VE-cadherin promoter and are required to mediate the transcriptional regulation of VE-cadherin by RA. These data indicate that RA may promote the expression of endothelial genes resulting in endothelial-like differentiation, or provide a mechanism whereby circulating endothelial progenitor cells could be incorporated into a growing organ or tumor.

Circulating tumor cells (CTCs) are rare cancer cells in the bloodstream thought to have a key role in cancer metastasis. There are enormous interests in their analysis for castration-resistant prostate cancer (CRPC) due to their tendency to metastasize to bone. In this study, we applied a surface marker-independent microfluidic device for CTCs capture and retrieval that is based on cell size and deformability. Our device was able to capture 90% and recover 30% of tumor cells in spike experiments. We also developed and validated assays capable of detecting the transcript levels of selected molecular markers in single cells prepared with C1TM Single-Cell AutoPrep System. Blood samples drawn from 34 CRPC patients and 10 primary prostate cancer patients (PPC) were preceded for CTC isolation and molecular characterization. We found that 73% CRPC patients and 10% PPC patients were positive for EpCAM, and only about half of the EpCAM-positive CRPC cases were PSA or AR positive. It is interesting that while there is a strong association between AR and PSA expression in CTC samples, there is no correlation between AR or PSA transcripts in CTCs and serum PSA protein levels in CRPC patients. The positive detection of AR or PSA, but not EpCAM is statistically associated with poor prognosis of the CRPC patients. Very intriguingly, we identified a novel androgen receptor (AR) transcript variant -ARv from four CRPC patients’ CTCs. This ARv lacks a 554bp sequence region in the ligand binding domain (LBD), resulting in a loss of the entire LBD. Clinical data analysis revealed that the ARv is significantly associated with worse survival of CRPC patients (p Citation Format: Zhigang Kang, Avani Shah, Yunkai Yu, Yuelin Zhu, Ali Asgar Bhagat, Kyra Zhao, Andrew Wu, James Gao, Ravi Madan, James Gulley, William Dahut, Paul Meltzer, Liang Cao. A novel transcript variant of androgen receptor identified in circulating tumor cells from castration-resistant prostate cancer patients as a potentially prognostic biomarker. [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 366. doi:10.1158/1538-7445.AM2015-366

Established human breast cancer cell lines are widely used as experimental models in breast cancer research. While these cell lines and their variants share many phenotypic characteristics with human breast tumors, the extent to which they reflect the underlying molecular biology of breast cancer remains controversial. We explored this issue using a probabilistic rather than heuristic approach. Data from gene expression microarrays were used to compare the global structures of the transcriptomes of three estrogen receptor alpha positive (ER+) human breast cancer cell lines (MCF-7, T47D, ZR-75-1) and 13 human breast tumors (11 ER+; 2 ER-). Linear representations of the respective data structures were obtained by deriving those top principal components (PCs) required to capture > or =80% of the cumulative variance for each data set (M PCs). We then identified those genes most highly correlated with the M PCs (Pearson's correlation coefficient r > or =0.800) and identified a group of 36 genes commonly correlated with both the cell line (M = 5 PCs) and tumor (M = 6 PCs) data structures. All 36 common genes were correlated with PC1 from the breast tumor data: 21/36 genes were correlated with PC1, 14/36 genes correlated with PC2, and 1/36 genes correlated with PC3 from the cell line data. Genes important in defining the data structures include NFkappaB p65, IGFBP-6, ornithine decarboxylase-1, and paxillin. When data from MDA-MB-435 xenografts (ER-) were included in the analysis, we were unable to find any common genes between these xenografts and the breast tumors. These data clearly imply that MCF-7, T47D, and ZR-75-1 cells and ER+ breast tumors share substantial global similarities in the structures of their respective transcriptomes, and that these cell lines are good models in which to identify molecular events that are likely to be important in some ER+ human breast cancers.

Circulating tumor cells (CTCs) are rare cancer cells circulating in the bloodstream that are thought to have a key role in cancer metastasis. They have been associated with worse prognosis in several major cancer types. There is an intense interest in the molecular analysis of CTC to gain information that would assist treatment decisions and potentially improve the disease outcome. There are enormous interests in the analysis of CTCs for castration-resistant prostate cancer (CRPC) due to the nature of the tumor that tends to metastasize to bone, making them generally inaccessible to obtain biopsies. In this study, we applied a surface marker independent, label-free cell trap microfluidics device for CTCs isolation and retrieval based on cell size and deformability. A total of 47 blood samples drawn from 36 patients with CRPC were preceded for CTC isolation followed by molecular characterization using epithelial tumor markers and prostate cancer specific markers. We identified a novel androgen receptor (AR) splice variant from four patients' CTCs, and confirmed by DNA sequencing that this variant lacks a 554bp region in the ligand binding domain of the receptor in comparison with wild type human AR. Patients with this splice variant were found to be associated with poor survival (p Note: This abstract was not presented at the meeting. Citation Format: Zhigang Kang, Yunkai Yu, Liang Cao, Paul S. Meltzer, Yuelin Zhu, Avan Shah, James Gulley, William Duht, Ravi Madan, Ali Bhagat, Kyra Zhao. A novel AR splice variant identified in circulating tumor cells from castration-resistant prostate cancer patient blood as a potentially prognostic biomarker. [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 4815. doi:10.1158/1538-7445.AM2014-4815

NUP98 gene fusions, created by non-random chromosomal translocations, are associated with a wide spectrum of high risk hematologic malignancies, and typically lead to overexpression of abdominal-b HOXA genes, a common theme shared by ∼50% of AML patients. We have generated a transgenic mouse model of the NUP98-PHF23 (NP23) gene fusion, initially identified in patients with AML, which develop AML, erythroleukemia, pre-T lymphoblastic leukemia (pre-T LBL), and a novel pre-B1 B cell acute lymphoblastic leukemia (pre-B1 ALL). A common theme in the leukemias and the premalignant hematopoietic tissues, is the overexpression of a Hoxa/b +Meis1 stem cell-like gene expression signature. GSEA analysis reveals this signature to be enriched in both human AML and ALL malignancies, and in human HSPC profiles. In addition, we found Bahcc1, a gene not previously associated with malignancy, to consistently segregate with the Hoxa/b+Meis1 signature in the NP23 leukemias and the premalignant tissues, independent of hematopoietic cell lineage. Furthermore, data-mining revealed BAHCC1 to be markedly overexpressed in AML patients with HOXA9/MEIS1 overexpression, and in a subset of MLL rearranged pre-B-ALL patients, suggesting BAHCC1 may be a previously unsuspected marker of leukemic transformation. NUP98-PHF23 belongs to a subset of fusion oncoproteins (including some MLL- and NUP98-fusions) that are potently tumorigenic and act by abrogating the normal reading, writing and erasure of histone methylation. Wild type PHF23 binds H3K4me3 residues via a PHD domain, therefore we used ChIP-seq to characterize global chromatin H3K4me3 and NP23 enrichment in NP23 leukemia derived cell lines. The vast majority (88%) of NP23 binding sites were enriched for H3K4me3 binding. Conversely, the NP23 protein co-localized at only 1.6% of all H3K4me3 enriched sites (including Hoxa, Hoxb and Meis1 loci) identifying these sites as direct targets of the NP23 fusion protein. Given that the NP23 fusion appears to function, at least in part, via binding to H3K4me3 sites at specific loci, we hypothesized that NP23 cells would be sensitive to disruption of the H3K4me4 binding by the NP23 PHD domain. Treatment of leukemic NP23 cells with Tetraethylthiuram disulfide (Disulfiram), a small molecule shown to inhibit PHD domain binding of H3K4me3 marks in vitro, rapidly and selectively killed NP23 myeloblasts but not control myeloblast cell lines (188G3, 189E6 and 32D) at 2 µM. Cell death was rapid, being 100% complete within 24 hours. Cell death was preceded by decreased levels of NP23 protein bound at target loci and decreased expression of these loci (e.g., Hoxa7/9/10, Hoxb5 and Meis1). We conclude that inhibitors of H3K4me3 PHD domain readers are promising therapeutic compounds that can kill leukemic cells driven by proteins that aberrantly read or write the histone code. The NP23 model provides a robust platform on which to identify and improve such compounds. Disclosures: Denu: Sirtris-GSK: Consultancy.

Abstract 2467 NUP98-fusions although rare, have been associated with de novo acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, T-cell acute lymphoblastic leukemia (T-ALL) and therapy-related myeloid malignancies. The NUP98-PHF23 (NP23) gene fusion was cloned from an acute myeloid leukemia (AML) patient with a t(11;17)(p15;p13) chromosome translocation. The nucleoporin 98 protein (NUP98), normally a component of the nuclear pore complex, is known to be fused to at least 28 different fusion partners as a result of structural chromosomal rearrangements associated with hematological malignancies. PHF23 encodes the Plant homeodomain (PHD) finger 23 protein. PHF23 is largely uncharacterized, but the PHD finger motif has been shown to act as a reader of di- and tri-methylated histone 3 lysine 4 (H3K4me2/3) marks. This suggests that PHF23 may function in chromatin regulation and that the NP23 fusion protein may play a role in aberrant chromatin modification at domains of active gene transcription. To determine the oncogenic potential of NP23, we generated a transgenic mouse model that expressed the human fusion gene in hematopoietic tissues. We have characterized two founder lines (C10 and B10) expressing the NP23 fusion in hematopoietic tissue. Most of the offspring from the C10 line developed an AML that closely resembled the human disease, with increased blasts in the blood or bone marrow, widespread organ infiltration, and myeloid immunophenotype. Onset of disease was as early as 4.5 months, and 70 percent of the NP23 mice succumbed to leukemia by 12 months of age. Of note, an independent line (B10) developed a wider spectrum of leukemias but with similar age of onset and penetrance. The B10 mice predominantly developed T-ALL and AML, and four cases of B-ALL and one erythroleukemia, indicating that the NP23 protein was oncogenic in several different hematopoietic cell types. AMLs typically demonstrated an aberrant Mac-1+/B220dim phenotype, which has previously been recognized in leukemias caused by overexpression of the Hoxa cluster genes Hoxa5,7,9,10,11. Microarray gene expression analysis identified the Hoxa cluster genes to be markedly overexpressed, and validation by RQ-PCR demonstrated that Hoxa5, a7, a9 and a10 overexpression ranged from 10- to greater than 1000-fold increased in both the AML and T-ALL samples compared to wild type hematopoietic tissues; these Hoxa cluster genes were also overexpressed in hematopoietic tissues from clinically healthy NP23 transgenic mice. We also identified a novel transcript, Gm525 (homologue of H. sapiens C17orf67), that is markedly (100x) elevated specifically in the T-ALL samples. Most T-ALL samples have HD or PEST domain Notch1 mutations, and Notch1 mRNA levels, as well as its downstream target Hes1, are elevated in the T-ALLs compared to WT thymus. Immortal cell lines were established from two of the NP23 T-ALLs, and ChIP-seq was used to assay the genome wide pattern of H3K4me3 and H3K27me3 histone marks. Results show abundant levels of H3K4me3 at the Hoxa locus which tightly correlates to the increased Hoxa cluster gene expression seen in the cell lines. The NP23 model will be useful for identifying oncoproteins involved in leukemic transformation, particularly those oncoproteins which play a role in chromatin modification or are downstream targets of the HOXA genes. Disclosures: No relevant conflicts of interest to declare.

miRNAs are ∼22nt long non-coding small RNAs that negatively regulate gene expression at the post-transcriptional level and play key roles in fundamental cellular processes. Deregulation of miRNAs has been observed in several diseases including cancer. Sarcomas are a heterogeneous group of rare mesenchymal malignancies with high rates of metastasis and local recurrence. A large variety of histotypes with very few reliable markers makes sarcoma diagnosis challenging. Hence, a refined classification scheme with novel diagnostic, prognostic and predictive markers would be clinically valuable. Additionally, tumor specific alterations in the miRNA expression profile may lead to the identification of miRNA targets important in sarcoma tumor biology. The diagnostic value of miRNA expression profiling was assessed in a mixed series of 161 sarcomas representing 12 histopathological subtypes, 5 normal smooth muscle, 4 normal skeletal muscle and 4 normal adipose tissues, using Agilent miRNA oligoarrays containing 470 human and 64 viral miRNAs. High-throughput miRNA sequencing (Illumina) was performed in a subset (n=10) of these tumors to validate results from the array-based expression profiling. An unsupervised cluster analysis performed on the 174 tumor and normal samples revealed groups based predominantly on diagnosis and tissue-type. All hemangiopericytomas and dermatofibrosarcomas, most rhabdomyosarcomas, Ewing's sarcomas, myxoid/round-cell liposarcomas, synovial sarcomas, leiomyosarcomas, osteosarcomas, normal smooth muscle, normal skeletal muscle and normal adipose tissue formed distinct clusters. About half of the pleomorphic/dedifferentiated/well-differentiated liposarcomas and fibrosarcomas and a quarter of the malignant peripheral nerve sheath tumors grouped together whereas most of the malignant fibrous histiocytomas clustered poorly. Discriminatory miRNAs were identified for the various subtypes (e.g., miR-146, miR-99 and miR-222 were highly expressed in the hemangiopericytomas, miR-375, miR-206 and miR-1 in the rhabdomyosarcomas, miR-181, miR-101 and miR-10 in the Ewing's sarcomas, miR-140-3p, miR-199 and miR-152 in osteosarcomas and miR-143, miR-145 and miR-28 in leiomyosarcomas). miRNAs that distinguished normal skeletal muscle from rhabdomysarcomas (e.g. miR-130, miR-133), normal smooth muscle from leiomyosarcomas (e.g. miR-142, miR-376) and normal adipose tissue from liposarcomas (e.g. miR-155, miR-365) were also identified. The results from sequencing were highly correlated with the results from the microarray analysis. Distinct miRNA expression profiles that correlate with lineage and tumor biology were identified not only in sarcomas with simple type-specific genetic alterations but also in the pleomorphic subtypes suggesting an important role for miRNAs in their tumorigenesis. 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 3024.