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PDF file - 57K, SuperArray Analysis of Oncogenes, Tumor Suppressors, and Stem Cell-Related Genes in MPM Lines Following Selective PRC2 Knockdown or 5 �M DZNep.

PDF file - 661K, TaqMan Primers, shRNA, and Antibodies.

PDF file - 102K, Effects of DZNep treatment on cultured mesothelioma cell lines relative to normal mesothelia cells. Pyrosequencing analysis of NBL2 DNA repeat in normal mesothelia (LP9) and MPM cell lines cultured in the presence or absence of DZNep.

PDf file - 63K, qRT-PCR and immunoblot analysis of EZH2 and EED levels in MPM lines following transduction with shRNA targeting EZH2, EED, or sham sequences. Effects of knock-down of EZH2 and EED on proliferation of MPM lines.

Effect of mithramycin on target gene promoters.

Genes Simultaneously Modulated in Cultured MES1 and MES7 Cells and Xenografts following Mithramycin Treatment.

PDF file - 63K, Genes Simultaneously Modulated by DZNep in MES1 and H28.

qRT-PCR analysis of differentiation-related genes

PDF file - 661K, Photomicrographs of NMES1 and NMES2 normal pleura cultures, and MES1 through MES4 mesothelioma cell lines established at the NCI.

Purpose: The DNA methylation paradox, manifested as derepression of cancer-testis antigens, and silencing of tumor suppressors during malignant transformation, provides the rationale for the utilization of chromatin remodeling agents for cancer therapy. A phase I trial was done to examine pharmacokinetics, toxicities, and gene expression mediated by 5-aza-2′-deoxycytidine (DAC) in patients with thoracic malignancies.Experimental Design: Thirty-five patients with cancers refractory to standard therapy received continuous 72-hour DAC infusions using a phase I dose-escalation schema. Each full course of therapy consisted of two identical 35-day cycles. Plasma DAC levels were evaluated by liquid chromatography-mass spectrometry techniques. Quantitative reverse transcription-PCR, methylation-specific PCR, and immunohistochemical techniques were used to evaluate NY-ESO-1, MAGE-3, and p16 expression in tumor biopsies. Long oligonucleotide arrays were used to evaluate gene expression profiles in laser-captured tumor cells before and after DAC exposure.Results: Thirty-five patients were evaluable for toxicities; 25 were evaluable for treatment response. Myelosuppression constituted dose-limiting toxicity. The maximum tolerated dose of DAC was 60 to 75 mg/m2 depending on the number of prior cytotoxic chemotherapy regimens. No objective responses were observed. Plasma DAC concentrations approximated thresholds for gene induction in cultured cancer cells. Target gene induction was observed in 36% of patients. Posttreatment antibodies to NY-ESO-1 were detected in three patients exhibiting NY-ESO-1 induction in their tumor tissues. Complex, heterogeneous gene expression profiles were observed in pretreatment and posttreatment tissues.Conclusion: Prolonged DAC infusions can modulate gene expression in primary thoracic malignancies. These findings support further evaluation of DNA-demethylating agents alone or in combination with other regimens targeting induced gene products for the treatment of these neoplasms.

PDF file - 97K, Supplementary Materials and Methods.

In this study, we generated induced pluripotent stem cells (iPSC) from normal human small airway epithelial cells (SAEC) to investigate epigenetic mechanisms of stemness and pluripotency in lung cancers. We documented key hallmarks of reprogramming in lung iPSCs (Lu-iPSC) that coincided with modulation of more than 15,000 genes relative to parental SAECs. Of particular novelty, we identified the PRC2-associated protein, ASXL3, which was markedly upregulated in Lu-iPSCs and small cell lung cancer (SCLC) lines and clinical specimens. ASXL3 overexpression correlated with increased genomic copy number in SCLC lines. ASXL3 silencing inhibited proliferation, clonogenicity, and teratoma formation by Lu-iPSCs, and diminished clonogenicity and malignant growth of SCLC cells in vivo. Collectively, our studies validate the utility of the Lu-iPSC model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and highlight ASXL3 as a novel candidate target for SCLC therapy. Cancer Res; 77(22); 6267–81. ©2017 AACR.

PDF file - 62K, Demographic Data Pertaining to the Patients from whom NCI-SB-MES 1-4 lines Were Established.

Supplementary Figure 1 from Phase I Study of Decitabine-Mediated Gene Expression in Patients with Cancers Involving the Lungs, Esophagus, or Pleura

PDF file - 245K, EZH2 expression in tumors correlates with cell line expression.

DNA Methylation Pattern of PRC2 Target Genes

Purpose: Specificity protein 1 (SP1) is an oncogenic transcription factor overexpressed in various human malignancies. This study sought to examine SP1 expression in malignant pleural mesotheliomas (MPM) and ascertain the potential efficacy of targeting SP1 in these neoplasms.Experimental Design: qRT-PCR, immunoblotting, and immunohistochemical techniques were used to evaluate SP1 expression in cultured MPM cells and MPM specimens and normal mesothelial cells/pleura. MTS, chemotaxis, soft agar, β-galactosidase, and Apo-BrdUrd techniques were used to assess proliferation, migration, clonogenicity, senescence, and apoptosis in MPM cells following SP1 knockdown, p53 overexpression, or mithramycin treatment. Murine subcutaneous and intraperitoneal xenograft models were used to examine effects of mithramycin on MPM growth in vivo. Microarray, qRT-PCR, immunoblotting, and chromatin immunoprecipitation techniques were used to examine gene expression profiles mediated by mithramycin and combined SP1 knockdown/p53 overexpression and correlate these changes with SP1 and p53 levels within target gene promoters.Results: MPM cells and tumors exhibited higher SP1 mRNA and protein levels relative to control cells/tissues. SP1 knockdown significantly inhibited proliferation, migration, and clonogenicity of MPM cells. Mithramycin depleted SP1 and activated p53, dramatically inhibiting proliferation and clonogenicity of MPM cells. Intraperitoneal mithramycin significantly inhibited growth of subcutaneous MPM xenografts and completely eradicated mesothelioma carcinomatosis in 75% of mice. Mithramycin modulated genes mediating oncogene signaling, cell-cycle regulation, senescence, and apoptosis in vitro and in vivo. The growth-inhibitory effects of mithramycin in MPM cells were recapitulated by combined SP1 knockdown/p53 overexpression.Conclusions: These findings provide preclinical rationale for phase II evaluation of mithramycin in patients with mesothelioma. Clin Cancer Res; 22(5); 1197–210. ©2015 AACR.

Purpose: Polycomb group (PcG) proteins are critical epigenetic mediators of stem cell pluripotency, which have been implicated in the pathogenesis of human cancers. This study was undertaken to examine the frequency and clinical relevance of PcG protein expression in malignant pleural mesotheliomas (MPM).Experimental Design: Microarray, quantitative reverse transcriptase PCR (qRT-PCR), immunoblot, and immunohistochemistry techniques were used to examine PcG protein expression in cultured MPM, mesothelioma specimens, and normal mesothelial cells. Lentiviral short hairpin RNA techniques were used to inhibit EZH2 and EED expression in MPM cells. Proliferation, migration, clonogenicity, and tumorigenicity of MPM cells either exhibiting knockdown of EZH2 or EED, or exposed to 3-deazaneplanocin A (DZNep), and respective controls were assessed by cell count, scratch and soft agar assays, and murine xenograft experiments. Microarray and qRT-PCR techniques were used to examine gene expression profiles mediated by knockdown of EZH2 or EED, or DZNep.Results:EZH2 and EED, which encode components of polycomb repressor complex-2 (PRC-2), were overexpressed in MPM lines relative to normal mesothelial cells. EZH2 was overexpressed in approximately 85% of MPMs compared with normal pleura, correlating with diminished patient survival. Overexpression of EZH2 coincided with decreased levels of miR-101 and miR-26a. Knockdown of EZH2 orEED, or DZNep treatment, decreased global H3K27Me3 levels, and significantly inhibited proliferation, migration, clonogenicity, and tumorigenicity of MPM cells. Common as well as differential gene expression profiles were observed following knockdown of PRC-2 members or DZNep treatment.Conclusions: Pharmacologic inhibition of PRC-2 expression/activity is a novel strategy for mesothelioma therapy. Clin Cancer Res; 18(1); 77–90. ©2011 AACR.

PDF file - 65K, Top 20 Gene Sets Enriched in MES1 and H28 Cells following DZNep Treatment.

PDF file - 54K, Top 20 Gene Ontologies Modulated by DZNep in MES1 and H28 Cells.

PDF file - 66K, Summary of percentages of mesothelioma or normal mesothelia specimens expressing EZH2 in figure 2D.

Supplementary Table 1 from Phase I Study of Decitabine-Mediated Gene Expression in Patients with Cancers Involving the Lungs, Esophagus, or Pleura

Brother of the Regulator of Imprinted Sites (BORIS) is a mammalian CTCF paralog with the same central 11Zn fingers (11ZF) that mediate specific interactions with varying ∼50-bp target sites. Regulated in vivo occupancy of such sites may yield structurally and functionally distinct CTCF/DNA complexes involved in various aspects of gene regulation, including epigenetic control of gene imprinting and X chromosome inactivation. The latter functions are mediated by meCpG-sensitive 11ZF binding. Because CTCF is normally present in all somatic cells, whereas BORIS is active only in CTCF- and 5-methylcytosine–deficient adult male germ cells, switching DNA occupancy from CTCF to BORIS was suggested to regulate site specificity and timing of epigenetic reprogramming. In addition to 11ZF-binding paternal imprinting control regions, cancer-testis gene promoters also undergo remethylation during CTCF/BORIS switching in germ cells. Only promoters of cancer testis genes are normally silenced in all somatic cells but activated during spermatogenesis when demethylated in BORIS-positive germ cells and are found aberrantly derepressed in various tumors. We show here that BORIS is also expressed in multiple cancers and is thus itself a cancer-testis gene and that conditional expression of BORIS in normal fibroblasts activates cancer-testis genes selectively. We tested if replacement of CTCF by BORIS on regulatory DNA occurs in vivo on activation of a prototype cancer-testis gene, MAGE-A1. Transition from a hypermethylated/silenced to a hypomethylated/activated status induced in normal cells by 5-aza-2′-deoxycytidine (5-azadC) was mimicked by conditional input of BORIS and is associated with complete switching from CTCF to BORIS occupancy at a single 11ZF target. This site manifested a novel type of CTCF/BORIS 11ZF binding insensitive to CpG methylation. Whereas 5-azadC induction of BORIS takes only few hours, derepression of MAGE-A1 occurred 1 to 2 days later, suggesting that BORIS mediates cancer-testis gene activation by 5-azadC. Indeed, infection of normal fibroblasts with anti-BORIS short hairpin RNA retroviruses before treatment with 5-azadC blocked reactivation of MAGE-A1. We suggest that BORIS is likely tethering epigenetic machinery to a novel class of CTCF/BORIS 11ZF target sequences that mediate induction of cancer-testis genes.

Supplementary Figure 2 from Conditional Expression of the CTCF-Paralogous Transcriptional Factor BORIS in Normal Cells Results in Demethylation and Derepression of MAGE-A1 and Reactivation of Other Cancer-Testis Genes

PDF file - 52K, ABCG2 Expression in Side Populations (sp) and Non-side Populations (nsp) of A549 and Calu-6 Cells Cultured with or without CSC

PDF file - 87K, Bar graph depicting total number of differentially expressed genes, as well as relative numbers induced or repressed under various treatment conditions

Limited information is available about epigenetic mechanisms by which cigarette smoke enhances the initiation and progression of lung cancer. To examine this issue, A549 and Calu-6 lung cancer cells were cultured in normal media with or without tobacco smoke condensate (TSC) under clinically relevant exposure conditions. Ten-day TSC exposure dramatically increased the tumorigenicity of lung cancer cells in nude mice. Microarray and quantitative reverse transcription-PCR (RT-PCR) experiments revealed that this phenomenon coincided with diminished expression of Dickkopf-1 (Dkk-1). Western blot, chromatin immunoprecipitation, methylation-specific PCR, and pyrosequencing experiments showed that repression of Dkk-1 coincided with decreased H4K16Ac, increased H3K27me3, and recruitment of SirT1, EZH2, SUZ12, and Bmi1 without DNA hypermethylation within the Dkk-1 promoter despite prolonged TSC exposures. Removal of TSC from culture media resulted in loss of promoter-associated polycomb repressor complexes and reexpression of Dkk-1. siRNA-mediated knockdown of EZH2 and SirT1 partially abrogated TSC-mediated inhibition of Dkk-1 expression. Western blot and quantitative RT-PCR array experiments showed that TSC exposure as well as knockdown of Dkk-1 activated Wnt signaling and significantly up-regulated Wnt5a in lung cancer cells. Knockdown of Dkk-1 recapitulated the dramatic protumorigenic effects of TSC exposure in Calu-6 cells. Despite the transient nature of Dkk-1 repression following TSC exposure in vitro, Dkk-1 remained silenced in tumor xenografts derived from TSC-treated Calu-6 cells. Collectively, these data provide evidence that cigarette smoke directly engages polycomb machinery to activate a signaling network implicated in maintenance of cancer stem cells. [Cancer Res 2009;69(8):3570–8]

PDF file - 116K, qRT-PCR analysis of ABCG2 expression in untreated NSCLC and SCLC lines relative to SAEC, and in NSCLC and SCLC lines following CSC exposure

PDF file - 111K

Supplementary Figure 1 from Tobacco Smoke Induces Polycomb-Mediated Repression of Dickkopf-1 in Lung Cancer Cells

Supplementary Tables 1-6 from Inhibition of Histone Lysine Methylation Enhances Cancer–Testis Antigen Expression in Lung Cancer Cells: Implications for Adoptive Immunotherapy of Cancer

Supplementary Table 1 and Figure Legends from Conditional Expression of the CTCF-Paralogous Transcriptional Factor BORIS in Normal Cells Results in Demethylation and Derepression of MAGE-A1 and Reactivation of Other Cancer-Testis Genes

Supplementary Figures 1 and 3 from Conditional Expression of the CTCF-Paralogous Transcriptional Factor BORIS in Normal Cells Results in Demethylation and Derepression of MAGE-A1 and Reactivation of Other Cancer-Testis Genes

PDF file - 363K, Ingenuity pathway analysis demonstrating two common networks targeted by mithramycin in lung cancer cells in vitro and in vivo

PDF file - 76K, TaqMan and ChIP Primers, and Antibodies

PDF file - 346K, Ingenuity pathway analysis depicting representative intracellular networks modulated by mithramycin lung cancer xenografts

The primate brain is equipped to learn and remember newly encountered visual stimuli such as faces and objects. In the macaque inferior temporal (IT) cortex, neurons mark the familiarity of a visual stimulus through response modification, often involving a decrease in spiking rate. Here, we investigate the emergence of this neural plasticity by longitudinally tracking IT neurons during several weeks of familiarization with face images. We found that most neurons in the anterior medial (AM) face patch exhibited a gradual decline in their late-phase visual responses to multiple stimuli. Individual neurons varied from days to weeks in their rates of plasticity, with time constants determined by the number of days of exposure rather than the cumulative number of presentations. We postulate that the sequential recruitment of neurons with experience-modified responses may provide an internal and graded measure of familiarity strength, which is a key mnemonic component of visual recognition.

Preclinical studies inform and guide the development of novel treatment combination strategies that bridge the laboratory with the clinic. We aimed to evaluate approaches cancer researchers used to justify advancing new combinations of molecularly targeted agents and radiation treatment into early-phase human clinical trials. Unsolicited early phase clinical trial proposals submitted to the National Cancer Institute's Cancer Therapy Evaluation Program between January 2016 and July 2020 were curated to quantify key characteristics and proportion of preclinical data provided by trialists seeking to conduct molecularly targeted agent-radiation combination studies in cancer patients. These data elucidate the current landscape for how the rationale for a molecularly targeted agent-radiation combination therapy is supported by preclinical research and illustrate unique challenges faced in translation at the intersection of precision medicine and radiation oncology.

Cilia are highly specialized organelles that extend from the cell membrane and function as cellular signaling hubs. Thus, cilia formation and the trafficking of signaling molecules into cilia are essential cellular processes. TULP3 and Tubby (TUB) are members of the tubby-like protein (TULP) family that regulate the ciliary trafficking of G-protein coupled receptors, but the functions of the remaining TULPs (i.e., TULP1 and TULP2) remain unclear. Herein, we explore whether these four structurally similar TULPs share a molecular function in ciliary protein trafficking. We found that TULP3 and TUB, but not TULP1 or TULP2, can rescue the defective cilia formation observed in TULP3-knockout (KO) hTERT RPE-1 cells. TULP3 and TUB also fully rescue the defective ciliary localization of ARL13B, INPP5E, and GPR161 in TULP3 KO RPE-1 cells, while TULP1 and TULP2 only mediate partial rescues. Furthermore, loss of TULP3 results in abnormal IFT140 localization, which can be fully rescued by TUB and partially rescued by TULP1 and TULP2. TUB’s capacity for binding IFT-A is essential for its role in cilia formation and ciliary protein trafficking in RPE-1 cells, whereas its capacity for PIP2 binding is required for proper cilia length and IFT140 localization. Finally, chimeric TULP1 containing the IFT-A binding domain of TULP3 fully rescues ciliary protein trafficking, but not cilia formation. Together, these two TULP domains play distinct roles in ciliary protein trafficking but are insufficient for cilia formation in RPE-1 cells. In addition, TULP1 and TULP2 play other unknown molecular roles that should be addressed in the future.

Radiopharmaceutical targeted therapy (RPT) has been studied for decades; however, recent clinical trials demonstrating efficacy have helped renewed interest in the modality. This article reviews National Cancer Institute (NCI)’s support of RPT through communication via workshops and interest groups, through funding extramural programs in academia and small business, and through intramural research, including preclinical and clinical studies. NCI has co-organized workshops and organized interest groups on RPT and RPT dosimetry to encourage the community and facilitate rigorous preclinical and clinical studies. NCI has been supporting RPT research through various mechanisms. Research has been funded through peer-reviewed NCI Research and Program Grants (RPG) and NCI Small Business Innovation Research (SBIR) Development Center, which funds small business-initiated projects, some of which have led to clinical trials. The NCI Cancer Therapy Evaluation Program (CTEP)’s Radiopharmaceutical Development Initiative supports RPT in NCI-funded clinical trials, including Imaging and Radiation Oncology Core (IROC) expertise in imaging QA and dosimetry procedures. Preclinical targeted a-emitter therapy (TAT) research at the NCI’s intramural program is ongoing, building on foundational work dating back to the 1980s. Ongoing “bench-to-bedside” efforts leverage the unique infrastructure of the National Institutes of Health’s (NIH) Clinical Center. Given the great potential of RPT, our goal is to continue to encourage its development that will generate the high-quality evidence needed to bring this multidisciplinary treatment to patients.

Primates readily learn new visual objects, with neurons in the inferior temporal cortex exhibiting diminished visual responses to familiar stimuli. The mechanisms of visual plasticity expressed within a neural population are largely unknown. Here we used chronic microwire electrodes in a face-selective cortical area in the macaque to longitudinally track cohorts of neurons across several weeks of exposure to novel faces. Neurons showed gradual adaptation in their late-phase visual responses, with cell-specific time constants ranging from two to twenty days. These time constants were governed by the number of testing days rather than by the cumulative number of stimulus exposures. This gradual buildup of altered visual responses may serve as an internal and graded mark of stimulus familiarity, a central component of visual recognition.

Genital and inverse psoriasis can develop in more than one-third of patients who have psoriasis. Psoriatic plaques in the genital and intertriginous skin are challenging to treat because the skin is thin and often occluded, making it more sensitive to certain therapies. Traditional guidelines indicate topical therapies, such as corticosteroids, topical calcineurin inhibitors (TCI), and vitamin D analogs as first-line recommendation in treating genital and inverse psoriasis. There have been developments in the treatment of genital and inverse psoriasis using systemic therapies, including IL-17 inhibitors and PDE-4 inhibitors.

Psoriasis is a chronic inflammatory condition for which eleven FDA-approved biologic therapies are approved. Over the past decade, studies have documented the higher efficacy of IL-17 and IL-23 inhibitors for the treatment of psoriasis compared to the TNF-alpha inhibitors and ustekinumab, an IL-12/23 inhibitor. Despite this, there remains an important role for the use of TNF-alpha inhibitors and ustekinumab in the treatment of psoriasis. Here, we review how considerations of infection and malignancy risk, patient demographics, treatment resistance, and comorbidities may make certain TNF-alpha inhibitors or ustekinumab an excellent choice for therapy in particular patient subgroups.

Metal electrodes with rough surfaces are often found to convert CO or CO2 to hydrocarbons and oxygenates with high selectivity and at high reaction rates in comparison with their smooth counterpart...

SARS-CoV-2 has changed global healthcare since the pandemic began in 2020. The safety of minimally invasive surgery (MIS) utilizing insufflation from the standpoint of safety to the operating room personnel is currently being explored. The aims of this guideline are to examine the existing evidence to provide guidance regarding MIS for the patient with, or suspecting of having, the SARS-CoV-2 as well as the healthcare team involved.Systematic literature reviews were conducted for 2 key questions (KQ) regarding the safety of MIS in the setting of COVID-19 pandemic. Reporting followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis criteria. Evidence-based recommendations were formulated using a narrative synthesis of the literature by subject experts. Recommendations for future research were also proposed.In KQ1, a total of 1361 articles were reviewed, with 2 articles meeting inclusion. In KQ2, a total of 977 articles were reviewed, with 4 articles met inclusions criteria, of which 2 studies reported on the SARS-CoV2 virus specifically. Despite many publications in the field, very little well-controlled and unbiased data exist to inform the recommendations. Of that which is available, it shows that both laparoscopic and open operations in Covid-positive patients had similar rates of OR staff positivity rates; however, patients who underwent laparoscopic procedures had a lower perioperative mortality than open procedures. Also, SARS-CoV-2 particles have been detected in the surgical plume at laparoscopy.With demonstrated equivalence of operating room staff exposure, and noninferiority of laparoscopic access with respect to mortality, either laparoscopic or open approaches to abdominal operations may be used in patients with SARS-CoV-2. Measures should be employed for all laparoscopic or open cases to prevent exposure of operating room staff to the surgical plume, as virus can be present in this plume.