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The canonical model of “small cell lung cancer” (SCLC) depicts tumors arising from dual inactivation of TP53 and RB1. However, many genomic studies have persistently identified tumors with no RB1 mutations. Here, we examined RB1 protein expression and function in SCLC. RB1 expression was examined by IHC analysis of 62 human SCLC tumors. These studies showed that ∼14% of SCLC tumors expressed abundant RB1 protein, which is associated with neuroendocrine gene expression and is enriched in YAP1 expression, but no other lineage proteins that stratify SCLC. SCLC cells and xenograft tumors with RB1 protein expression were sensitive to growth inhibition by the CDK4/6 inhibitor palbociclib, and this inhibition was shown to be dependent on RB1 expression by CRISPR knockout. Furthermore, a patient with biopsy-validated wild-type RB1 SCLC who received the CDK4/6 inhibitor abemaciclib demonstrated a dramatic decrease in mutant TP53 ctDNA allelic fraction from 62.1% to 0.4% and decreased tumor mass on CT scans. Importantly, IHC of the diagnostic biopsy specimen showed RB1 positivity. Finally, we identified a transcriptomics-based RB1 loss-of-function signature that discriminates between SCLC cells with or without RB1 protein expression and validated it in the patient who was responsive to abemaciclib, suggesting its potential use to predict CDK4/6 inhibitor response in patients with SCLC. Our study demonstrates that RB1 protein is an actionable target in a subgroup of SCLC, a cancer that exhibits no currently targetable mutations.

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Nutrient-deprived conditions in the tumor microenvironment (TME) restrain cancer cell viability due to increased free radicals and reduced energy production. In pancreatic cancer cells a cytosolic metabolic enzyme, wild-type isocitrate dehydrogenase 1 (wtIDH1), enables adaptation to these conditions. Under nutrient starvation, wtIDH1 oxidizes isocitrate to generate α-ketoglutarate (αKG) for anaplerosis and NADPH to support antioxidant defense. In this study, we show that allosteric inhibitors of mutant IDH1 (mIDH1) are potent wtIDH1 inhibitors under conditions present in the TME. We demonstrate that low magnesium levels facilitate allosteric inhibition of wtIDH1, which is lethal to cancer cells when nutrients are limited. Furthermore, the Food & Drug Administration (FDA)-approved mIDH1 inhibitor ivosidenib (AG-120) dramatically inhibited tumor growth in preclinical models of pancreatic cancer, highlighting this approach as a potential therapeutic strategy against wild-type IDH1 cancers.

Adenosine triphosphate-binding cassette (ABC) transporters, such as multidrug resistance protein 1 (MRP1), protect against cellular toxicity by exporting xenobiotic compounds across the plasma membrane. However, constitutive MRP1 function hinders drug delivery across the blood–brain barrier, and MRP1 overexpression in certain cancers leads to acquired multidrug resistance and chemotherapy failure. Small-molecule inhibitors have the potential to block substrate transport, but few show specificity for MRP1. Here we identify a macrocyclic peptide, named CPI1, which inhibits MRP1 with nanomolar potency but shows minimal inhibition of a related multidrug transporter P-glycoprotein. A cryoelectron microscopy (cryo-EM) structure at 3.27 Å resolution shows that CPI1 binds MRP1 at the same location as the physiological substrate leukotriene C4 (LTC 4 ). Residues that interact with both ligands contain large, flexible sidechains that can form a variety of interactions, revealing how MRP1 recognizes multiple structurally unrelated molecules. CPI1 binding prevents the conformational changes necessary for adenosine triphosphate (ATP) hydrolysis and substrate transport, suggesting it may have potential as a therapeutic candidate.

High‐grade neuroendocrine carcinoma of gynecologic origin (NEC‐GYN) is a highly aggressive cancer that often affects young women. The clinical management of NEC‐GYN is typically extrapolated from its counterpart, small cell carcinoma of the lung (SCLC), but, unfortunately, available therapies have limited benefit. In our NEC‐GYN cohort, median progression‐free survival (PFS) and overall survival (OS) were 1 and 12 months, respectively, indicating the highly lethal nature of this cancer. Our comprehensive genomic analyses unveiled that NEC‐GYN harbors a higher mutational burden with distinct mutational landscapes from SCLC. We identified 14 cancer driver genes, including the most frequently altered KMT2C (100%), KNL1 (100%), NCOR2 (100%), and CCDC6 (93%) genes. Transcriptomic analysis identified several novel gene fusions; astonishingly, the MALAT1 lincRNA gene was found in ˜ 20% of all fusion events in NEC‐GYN. Furthermore, NEC‐GYN exhibited a highly immunosuppressive state, intact RB1 expression, and was uniquely enriched with the YAP1 high molecular subtype. Our study identifies several potential therapeutic targets and suggests an urgent need to re‐evaluate the treatment options for NEC‐GYN., The neuroendocrine carcinoma of gynecologic origin (NEC‐GYN) is a deadly cancer that often affects young women. Treatment of NEC‐GYN is typically extrapolated from small cell lung cancer (SCLC), but, unfortunately, available treatments are inadequate. We report that NEC‐GYN is genomically distinct from SCLC, identify several potential therapeutic targets, and suggest an urgent need to re‐evaluate the treatment options for NEC‐GYN.

Responses of cells to signals are increasingly discovered to involve the binding of sequence-specific transcription factors outside of known target genes. We wanted to determine to what extent the genome-wide binding and function of a transcription factor are shaped by the cell type versus the stimulus. To do so, we induced the Heat Shock Response pathway in two distant cell lines with two different stimuli and related the binding of its master regulator HSF1 to nascent RNA and chromatin accessibility. We show that HSF1 binding patterns robustly retain their identity under different magnitudes of activation so that common HSF1 binding is globally associated with stimulus-specific transcription outcomes. HSF1-induced increase in DNA accessibility was modest in scale but occurred predominantly at remote genomic sites. Apart from regulating transcription at existing elements including promoters and enhancers, responses to heat shock may directly engage inactive chromatin.

Germline mutation of PTEN is causally observed in Cowden syndrome (CS) and is one of the most common, penetrant risk genes for autism spectrum disorder (ASD). However, the majority of individuals who present with CS-like clinical features are PTEN-mutation negative. Reassessment of PTEN promoter regulation may help explain abnormal PTEN dosage, as only the minimal promoter and coding regions are currently included in diagnostic PTEN mutation analysis. Therefore, we reanalyzed the architecture of the PTEN promoter using next-generation sequencing datasets. Specifically, run-on sequencing assays identified two additional transcription start regions (TSRs) at −2053 and −1906 basepairs from the canonical start of PTEN, thus extending the PTEN 5'UTR and redefining the PTEN promoter. We show that these novel upstream TSRs are active in cancer cell lines, human cancer and normal tissue. Furthermore, these TSRs can produce novel PTEN transcripts due to the introduction of new splice donors at −2041, −1826 and −1355, which may allow for splicing out of the PTEN 5'UTR or the first and second exon in upstream-initiated transcripts. Combining ENCODE ChIP-seq and pertinent literature, we also compile and analyze all transcription factors (TFs) binding at the redefined PTEN locus. Enrichment analyses suggest that TFs bind specifically to the upstream TSRs may be implicated in inflammatory processes. Altogether, these data redefine the architecture of the PTEN promoter, an important step toward a comprehensive model of PTEN transcription regulation, a basis for future investigations into the new promoters’ role in disease pathogenesis.

PURPOSE Immune checkpoint blockade (ICB) in conjunction with chemotherapy is approved for the treatment of extensive-stage small-cell lung cancer (SCLC). Although specific genomic abnormalities such as KEAP1 and STK11 gene mutations are associated with resistance to ICB in non-SCLC, no genomic abnormality has been found in association with resistance to ICB in SCLC. MATERIALS AND METHODS We first analyzed a retrospective cohort of 42 patients with SCLC treated with single-agent ICB or ICB combination (data set A). We then validated our results in a large prospective clinical trial of 460 patients (CheckMate 032, data set B). DNA and RNA sequencing were performed. RESULTS In data set A, patients treated with ICB with RB1 wild-type (WT) had a median overall survival (OS) of 23.1 months (95% CI, 9 to 37.5), whereas the RB1 mutant OS was 5 months (95% CI, 2.5 to 26; P = .04). Differentially expressed gene analysis between RB1 mutant and RB1 WT samples indicated the enrichment of downregulated immune-related genes and an immune exclusion phenotype among RB1 mutant but not in the RB1 WT tumor samples. We then assessed results from 460 patients enrolled in CheckMate 032, a trial of nivolumab (NIVO) or NIVO + ipilimumab only in SCLC. In this large cohort, RB1 WT patients had significantly improved outcome with NIVO therapy compared with mutant patients (hazard ratio, 1.41; 95% CI, 1.02 to 2.01; P = .041). High RB1 loss-of-function (LOF) signature scores significantly associated with neuroendocrine subtypes (ASCL1 and NeuroD1). However, neuroendocrine subtypes did not associate with OS. Remarkably, patients with lower RB1 LOF scores had longer OS following treatment with NIVO. CONCLUSION SCLC patients with RB1 WT status or lower RB1 LOF signature scores by transcriptomics have better outcomes with ICB monotherapy.

Das neuroendokrine Karzinom der Cervix uteri ist eine seltene und aggressive Form von Gebärmutterhalskrebs, die durch häufige Metastasierung schon bei Diagnosestellung und durch hohe Rezidivraten gekennzeichnet ist. Die Primärtherapie ist multimodal und beinhaltet oft eine Chemotherapie, zum Teil in Verbindung mit einer Strahlentherapie. Es liegen keine Daten vor, die Orientierung für das Vorgehen bei Rezidiven geben; Zweitlinientherapien werden aus Daten zum kleinzelligen Bronchialkarzinom abgeleitet. Die engmaschige Überwachung dieser Patientinnen im Hinblick auf Rezidive ist von größter Bedeutung. Dafür stellt die Bestimmung der zirkulierenden Tumor-DNA (ctDNA) im peripheren Blut eine attraktive, weil nicht-invasive Option dar. Mit dem ULP-WGS-Verfahren (ultra-low-pass whole-genome sequencing) lassen sich die Tumorlast und das Tumoransprechen beurteilen und Prognosen zur Rezidivierung abgeben; allerdings mangelt es bisher noch an Daten zur Rolle der ULP-WGS beim kleinzelligen Zervixkarzinom. Die vorliegende Arbeit stellt eine Patientin vor, deren Chemotherapie-Ansprechen und Tumor-Rezidivierung durch die ctDNA-Analyse korrekt überwacht wurden, wie radiologische Befunde bestätigten.

SummaryPancreatic cancer (Pancreatic Ductal Adenocarcinoma; PDAC) is highly resistant to chemotherapy. Effective alternative therapies have yet to emerge, leaving chemotherapy as the best available systematic treatment. The discovery of safe and available adjuncts that improve chemotherapeutic efficacy would potentially improve survival outcomes. We show that a hyperglycemic state enhances the efficacy of conventional single- and multi-agent chemotherapies against PDAC. Molecular analyses of tumors exposed to relatively high glucose levels revealed that a key metabolic pathway, glutathione biosynthesis, is diminished and underlies chemo-sensitization by enhancing oxidative injury to cancer cells. Inhibition of this pathway under normal conditions phenocopied a hyperglycemic state by enhancing chemotherapeutic efficacy in mouse PDAC, while rescuing the pathway under high glucose abrogated the anti-tumor effects observed with chemotherapy.

RUNX1T1 is highly expressed in ‘combined’ and ‘pure’ SCLC via genetic amplification or greater transcription, respectively. RUNX1T1 interacts with the CDKN1A (p21) promoter and is associated with reduced histone H3 acetylation, resulting in decreased p21 expression and increased E2F transcriptional activity in lung cancer cell lines. Thus, RUNX1T1 is both a potential marker and epigenetic modifier in SCLC., Small‐cell lung cancer (SCLC) can be subgrouped into common ‘pure’ and rare ‘combined’ SCLC (c‐SCLC). c‐SCLC features a mixed tumor histology of both SCLC and non–small‐cell lung cancer (NSCLC). We performed targeted exome sequencing on 90 patients with SCLC, including two with c‐SCLC, and discovered RUNX1T1 amplification specific to small cell tumors of both patients with c‐SCLC, but in only 2 of 88 ‘pure’ SCLC patients. RUNX1T1 was first identified in the fusion transcript AML1/ETO, which occurs in 12%‐15% of acute myelogenous leukemia (AML). We further show higher expression of RUNX1T1 in the SCLC component of another c‐SCLC tumor by in situ hybridization. RUNX1T1 expression was enriched in SCLC compared with all other cancers, including NSCLC, in both cell lines and tumor specimens, as shown by mRNA level and western blotting. Transcriptomic analysis of hallmark genes decreased by stable RUNX1T1 overexpression revealed a significant change in E2F targets. Validation experiments in multiple lung cancer cell lines showed that RUNX1T1 overexpression consistently decreased CDKN1A (p21) expression and increased E2F transcriptional activity, which is commonly altered in SCLC. Chromatin immunoprecipitation (ChIP) in these overexpressing cells demonstrated that RUNX1T1 interacts with the CDKN1A (p21) promoter region, which displayed parallel reductions in histone 3 acetylation. Furthermore, reduced p21 expression could be dramatically restored by HDAC inhibition using Trichostatin A. Reanalysis of ChIP‐seq data in Kasumi‐1 AML cells showed that knockdown of the RUNX1T1 fusion protein was associated with increased global acetylation, including the CDKN1A (p21) promoter. Thus, our study identifies RUNX1T1 as a biomarker and potential epigenetic regulator of SCLC.

PTEN is implicated in a wide variety of pathophysiological conditions and traditionally studied in the context of the PIK3–AKT–mTOR axis. Recent studies from our group and others have reported a novel role of PTEN in the regulation of transcription at the genome-wide scale. This emerging role of PTEN on global transcriptional regulation is providing a better understanding of various diseases, including cancer. Because cancer progression is an energy-demanding process and PTEN is known to regulate metabolic processes, we sought to understand the role of PTEN in transcriptional regulation under metabolic stress, a condition often developing in the tumor microenvironment. In the present study, we demonstrate that PTEN modulates genome-wide RNA Polymerase II occupancy in cells undergoing glucose deprivation. The glucose-deprived PTEN null cells were found to continue global gene transcription, which may activate a survival mode. However, cells with constitutive PTEN expression slow transcription, an evolutionary mechanism that may save cellular energy and activate programmed cell death pathways, in the absence of glucose. Interestingly, alternative exon usage by PTEN null cells is increased under metabolic stress in contrast to PTEN-expressing cells. Overall, our study demonstrates distinct mechanisms involved in PTEN-dependent genome-wide transcriptional control under metabolic stress. Our findings provide a new insight in understanding tumor pathology and how PTEN loss of function, whether by genetic or non-genetic mechanisms, can contribute to a favorable transcriptional program employed by tumor cells to escape apoptosis, hence developing more aggressive and metastatic phenotypes.

Maspin repression is frequently observed in prostate cancer; however, the molecular mechanism(s) causing the loss is not completely understood. Here, we demonstrate that inhibition of class I histone deacetylases (HDACs) mediates re-expression of maspin which plays an essential role in suppressing proliferation and migration capability in prostate cancer cells. Human prostate cancer LNCaP and DU145 cells treated with HDAC inhibitors, sodium butyrate, and trichostatin A, resulted in maspin re-expression. Interestingly, an exploration into the molecular mechanisms demonstrates that maspin repression in prostate tumor and human prostate cancer cell lines occurs via epigenetic silencing through an increase in HDAC activity/expression, independent of promoter DNA hypermethylation. Furthermore, transcriptional activation of maspin was accompanied with the suppression of HDAC1 and HDAC8 with significant p53 enrichment at the maspin promoter associated with an increase in histone H3/H4 acetylation. Our results provide evidence of maspin induction as a critical epigenetic event altered by class I HDACs in the restoration of balance to delay proliferation and migration ability of prostate cancer cells.

Exosomes are 30 to 150 nm-diameter lipid bilayer-enclosed extracellular vesicles that enable cell-to-cell communication through secretion and uptake. The exosomal cargoes contain RNA, lipids, proteins, and metabolites which can be delivered to recipient cells in vivo. In a healthy lung, exosomes facilitate interaction between adaptive and innate immunity and help maintain normal lung physiology. However, tumor-derived exosomes in lung cancer (LC) can, on the other hand, restrict immune cell proliferation, cause apoptosis in activated CD8+ T effector cells, reduce natural killer cell activity, obstruct monocyte differentiation, and promote proliferation of myeloid-derived suppressor and regulatory T cells. In addition, exosomes in the tumor microenvironment may also play a critical role in cancer progression and the development of drug resistance. In this review, we aim to comprehensively examine the current updates on the role of exosomes in lung carcinogenesis and their potential application as a diagnostic, prognostic, and therapeutic tool in lung cancer.

∼90% metastatic pancreatic ductal adenocarcinoma (mPDAC) occurs in the liver, and the 5-year survival rate for patients with mPDAC is only at 3%. We previously reported that liver endothelial cells (ECs) secreted soluble factors to promote colorectal cancer cell survival in a paracrine fashion. However, the effects of liver ECs on mPDAC have not been elucidated. In this study, we used primary ECs from non-neoplastic liver tissues. We treated PDAC cells with conditioned medium (CM) from liver ECs, with CM from PDAC as controls, and determined that liver EC-secreted factors increased PDAC cell growth. Using an unbiased receptor tyrosine kinase array, we identified human epidermal growth factor receptor 3 (HER3, also known as ErbB3) as a key mediator in EC-induced growth in PDAC cells that have HER3 expression (HER3 +ve). We found that EC-secreted neuregulins activated the HER3-AKT signaling axis, and that depleting neuregulins from EC CM or blocking HER3 with an antibody, seribantumab, attenuated EC-induced proliferation in HER3 +ve PDAC cells, but not in cells without HER3 expression. Furthermore, we determined that EC CM increased PDAC xenograft growth in vivo, and that seribantumab blocked EC-induced growth in xenografts with HER3 expression. These findings elucidated a paracrine role of liver ECs in promoting PDAC cell growth, and identified the HER3-AKT axis as a key mediator in EC-induced functions in PDAC cells with HER3 expression.Implicationsover 70% mPDAC express HER3. This study suggests the potential of using HER3-targeted therapies for treating patients with HER3 +ve mPDAC.

Gene transcription is a highly complex and strictly regulated process. RNA polymerase II (Pol II) C-terminal domain (CTD) undergoes massive cycles of phosphorylation and dephosphorylation during the process of gene transcription. These post-translational modifications of CTD provide an interactive platform for various factors required for transcription initiation, elongation, termination, and co-transcriptional RNA processing. Pol II CTD kinases and phosphatases are key regulators and any deviation may cause genome-wide transcriptional dysregulation leading to various pathological conditions including cancer. PTEN, a well known tumor suppressor, is one of the most commonly somatically altered in diverse malignancies. When mutated in the germline, PTEN causes cancer predisposition. Numerous studies have demonstrated that PTEN regulates the expression of hundreds of genes, however, no mechanism is known so far. PTEN is a dual specificity phosphatase, using both lipid and protein as substrates. In the present study, we demonstrate that PTEN interacts with the RNA Pol II and that PTEN expression is inversely correlated with global phosphorylation of Pol II CTD. Furthermore, PTEN dephosphorylates Pol II CTD in vitro with a significant specificity for Ser5p. Interestingly, ChIP-seq data analysis revealed that PTEN globally binds to promoter proximal regions, and PTEN loss increases genome-wide Pol II Ser5p occupancy, suggest that PTEN is a Pol II CTD phosphatase. Our observations demonstrate an unexplored function of PTEN with the potential of global transcriptional regulation, adding a new dimension to somatic carcinogenesis and germline cancer predisposition.

Control of gene expression is one of the most complex yet continuous physiological processes impacting cellular homeostasis. RNA polymerase II (Pol II) transcription is tightly regulated at promoter-proximal regions by intricate dynamic processes including Pol II pausing, release into elongation and premature termination. Pol II pausing is a phenomenon where Pol II complex pauses within 30–60 nucleotides after initiating the transcription. Negative elongation factor (NELF) and DRB sensitivity inducing factor (DSIF) contribute in the establishment of Pol II pausing, and positive transcription elongation factor b releases (P-TEFb) paused complex after phosphorylating DSIF that leads to dissociation of NELF. Pol II pausing is observed in most expressed genes across the metazoan. The precise role of Pol II pausing is not well understood; however, it’s required for integration of signals for gene regulation. In the present study, we investigated the role of phosphatase and tensin homolog (PTEN) in genome-wide transcriptional regulation using PTEN overexpression and PTEN knock-down models. Here we identify that PTEN alters the expression of hundreds of genes, and its restoration establishes genome-wide Pol II promoter-proximal pausing in PTEN null cells. Furthermore, PTEN re-distributes Pol II occupancy across the genome and possibly impacts Pol II pause duration, release and elongation rate in order to enable precise gene regulation at the genome-wide scale. Our observations demonstrate an imperative role of PTEN in global transcriptional regulation that will provide a new direction to understand PTEN-associated pathologies and its management.

Rapid surface functionalization of nanomaterials using covalent linkage following "green chemistry" remains challenging, and the quest for developing simple protocols is persisting. We report a nanomechanical microfluidic approach for the coupling of allenamide functionalized organic derivatives on the surface of thiol-modified silica nanoparticles using allenamide-thiol chemistry. The coupling principle involves the use of a microfluidic surface acoustic wave device that generates acoustic streaming-based chaotic fluid micromixing that enables mixing of laterally flowing fluids containing active components. This approach was used to demonstrate the direct surface labeling of thiol-modified silica nanoparticles using a selected group of modified fluorescent tags containing allenamide handles and achieved a total labeling efficiency of 83-90%. This green approach enabled a highly efficient surface functionalization under aqueous conditions, with tunable control over the conjugation process via the applied field. The dye-labeled silica particles were characterized using various analytical techniques and found to be biocompatible with potential in live cell bioimaging. It is envisaged that this bioconjugation strategy will find numerous applications in the field of bioimaging and drug delivery. S.S. and C.T.L. acknowledge support from the Biomedical Institute for Global Health Research & Technology (BIGHEART), National University of Singapore.

Germline mutation of PTEN is causally observed in Cowden syndrome (CS) and is one of the most common genetic causes of autism spectrum disorder (ASD). However, the majority of individuals who present with CS-like clinical features are found to be PTEN-mutation negative. Reassessment of PTEN promoter regulation may help explain abnormal PTEN dosage, as only the minimal promoter and coding regions are currently included in diagnostic PTEN mutation analysis. We reanalyzed the architecture of the PTEN promoter using next-generation sequencing datasets. Specifically, run-on sequencing assays identified two additional TSRs at −2052 and −1907 basepairs from the start of PTEN, thus redefining the PTEN promoter and extending the PTEN 5’UTR. The upstream TSRs described are active in cancer cell lines and human cancer and normal tissue. Further, these TSRs can produce novel PTEN transcripts due to the introduction of new splice sites. Evaluation of transcription factor binding specific to the upstream TSRs shows overrepresentation of TFs involved in inflammatory processes. Together, these data suggest that potentially clinically relevant promoter variants upstream of the known promoter may be overlooked in indivduals considered PTEN germline mutation-negative and may also explain lack of PTEN expression in sporadic neoplasias without PTEN somatic structural defects.

The lung microbiome plays an essential role in maintaining healthy lung function, including host immune homeostasis. Lung microbial dysbiosis or disruption of the gut-lung axis can contribute to lung carcinogenesis by causing DNA damage, inducing genomic instability, or altering the host's susceptibility to carcinogenic insults. Thus far, most studies have reported the association of microbial composition in lung cancer. Mechanistic studies describing host-microbe interactions in promoting lung carcinogenesis are limited. Considering cancer as a multifaceted disease where epigenetic dysregulation plays a critical role, epigenetic modifying potentials of microbial metabolites and toxins and their roles in lung tumorigenesis are not well studied. The current review explains microbial dysbiosis and epigenetic aberrations in lung cancer and potential therapeutic opportunities.

Display technology platforms offer their own unique set of challenges for chemical transformations, at the heart of which lies peptide macrocyclization. The amenable reactions for peptide macrocyclization on this platform need to meet a number of criteria like high reactivity, selectivity, mild conditions, irreversibility, and in many cases, a unique requirement to be assimilated into the translation machinery. Skillful utilization of these reactions has led to the formation of huge macrocyclic peptide libraries with varied linkages and topographies which have in turn led to the discovery of a number of hits for purposes such as drug discovery and others. Herein, we review those reactions which have mainly been applied in mRNA and phage display and discuss their technical characteristics and significance.

Neuroendocrine carcinoma of the cervix is a rare and aggressive form of cervical cancer that presents with frequent metastasis at diagnosis and high recurrence rates. Primary treatment is multimodal, which often includes chemotherapy with or without radiation therapy. There is no data available to guide treatment for recurrence, and second-line therapies are extrapolated from small cell lung carcinoma data. Close monitoring of these patients for recurrence is paramount. Evaluation of circulating tumor DNA (ctDNA) in the peripheral blood is an attractive approach due to its noninvasive nature. Ultra-low pass whole genome sequencing (ULP-WGS) can assess tumor burden, response to therapy, and predict recurrence; however, data are lacking regarding the role of ULP-WGS in small cell carcinoma of the cervix. This study demonstrates a patient whose response to chemotherapy and cancer recurrence was accurately monitored by ctDNA using ULP-WGS testing and confirmed with radiologic imaging findings.

6093 Background: High-grade neuroendocrine carcinoma (NEC) of the cervix and other gynecologic origins are rare and aggressive cancers that unfortunately affect young women with high mortality. Treatment recommendations are often extrapolated from their counterpart, small cell carcinoma of the lung (SCLC). In the present study, we have performed comprehensive genomic and transcriptomic analyses in a cohort of patients with NEC of the cervix and other gynecologic origins and compared them to SCLC. Methods: We have identified 27 patients diagnosed with NEC of gynecologic origin from 1998-2019. Of them, we were able to obtain archival tissue from 14 patients (17 samples), including seven cervical NEC, three ovarian NEC, and two endometrial NEC. Two ovarian and one cervical patient have recurrent tumors. Whole exome sequencing (WES) and RNA-seq were successfully performed in 14 and 13 samples, respectively. BWA-MEM was used for mapping (hg38), and GATK Haplotype Caller was used for WES analysis. Ensemble-VEP was used for variant annotation, and SIFT and PolyPhen-2 were used for pathogenic prediction of missense mutations. Stranded pair-end RNA-seq data were analyzed using STAR and DESeq2. SCLC data from cBioPortal and EBI (accession number EGAS00001000334) were used to compare mutation and transcriptomic profiles. Results: We found that TP53 is not mutated in our cohort, and RB1 is mutated only in 1 (7%) tumor. TP53 and RB1 are the most frequently mutated genes in small cell lung carcinoma. However, LRP1B, the third most mutated gene in SCLC was also mutated in 4 (29%) tumors. We observed that WNT, RTK-RAS, NOTCH, and MYC are among the top mutated pathways in our cohort. Among the top 20 mutated genes, only four genes, MUC4, KMT2C, MAP3K1, and HLA-A, were common in cervical, ovarian, and endometrial tumors suggesting a high diversity within the NEC of gynecologic origin. Our RNA-seq analysis revealed a distinct transcriptional signature when compared to SCLC, especially the expression pattern of SCLC molecular subtypes defining ASCL1, NEUROD1, POU3F2, and YAP1 genes. Surprisingly, we observed a high expression of the YAP1 gene in all of the 13 tumors. However, the YAP1+ subtype represents a minority in SCLC and known to predict chemotherapy resistance and lower survival. Conclusions: Our results suggest a unique mutational profile and transcriptional signature that are distinct compared to SCLC tumors. Therefore, there is an urgent need to re-evaluate the therapeutic options and targets for NEC of gynecologic origin.

While a role of promoter-proximal RNA Polymerase II (Pol II) pausing in regulation of eukaryotic gene expression is implied, the mechanisms and dynamics of this process are poorly understood. We performed genome-wide analysis of short capped RNAs (scRNAs) and Pol II chromatin immunoprecipitation sequencing (ChIP-seq) in human breast cancer MCF-7 cells to better understand Pol II pausing (Samarakkody, A., Abbas, A., Scheidegger, A., Warns, J., Nnoli, O., Jokinen, B., Zarns, K., Kubat, B., Dhasarathy, A. and Nechaev, S. (2015) RNA polymerase II pausing can be retained or acquired during activation of genes involved in the epithelial to mesenchymal transition. Nucleic Acids Res 43, 3938–3949). The data are available at the NCBI Gene Expression Omnibus under accession number GSE67041. For both ChIP and scRNA samples, we used paired end sequencing on the Illumina MiSeq instrument. For ChIP-seq, the use of paired end sequencing allowed us to avoid ambiguities in center-read definition. For scRNA seq, this allowed us to identify both the 5′-end and the 3′-end in the same run that represent, respectively, the transcription start sites and the locations of Pol II pausing. The sharpening of Pol II ChIP-seq metagene profiles when aligned against 5′-ends of scRNAs indicates that these RNAs can be used to define the start sites for the majority of mRNA transcription events.

Promoter-proximal RNA polymerase II (Pol II) pausing is implicated in the regulation of gene transcription. However, the mechanisms of pausing including its dynamics during transcriptional responses remain to be fully understood. We performed global analysis of short capped RNAs and Pol II Chromatin Immunoprecipitation sequencing in MCF-7 breast cancer cells to map Pol II pausing across the genome, and used permanganate footprinting to specifically follow pausing during transcriptional activation of several genes involved in the epithelial to mesenchymal transition (EMT). We find that the gene for EMT master regulator Snail (SNAI1), but not Slug (SNAI2), shows evidence of Pol II pausing before activation. Transcriptional activation of the paused SNAI1 gene is accompanied by a further increase in Pol II pausing signal, whereas activation of non-paused SNAI2 gene results in the acquisition of a typical pausing signature. The increase in pausing signal reflects increased transcription initiation without changes in Pol II pausing. Activation of the heat shock HSP70 gene involves pausing release that speeds up Pol II turnover, but does not change pausing location. We suggest that Pol II pausing is retained during transcriptional activation and can further undergo regulated release in a signal-specific manner.

Prostate cancer is a disease that is greatly affected by lifestyle, particularly diet, and is more prevalent in US and European countries compared with South and East Asia. Among several known causes and risk factors, nutrition plays an important role in prostate cancer pathogenesis. Various dietary components including polyphenols have been shown to possess anticancer properties. Dietary polyphenols have been the subject of extensive studies for the last decade because of their anticancer and chemopreventive potentials. Besides possessing various antitumor properties, dietary polyphenols also contribute to epigenetic changes associated with the fate of cancer cells and have emerged as potential drugs for therapeutic intervention. Various polyphenols have been shown to affect DNA methylation, histone posttranslational modifications, and microRNA expression patterns in prostate cancer. In this review, we discuss the contribution of dietary polyphenols to various epigenetic modifications in prostate cancer. Since prostate cancer and diet are intimately associated, polyphenol-rich diets that epigenetically modify tumor biology have great significance in the prevention and management of prostate cancer.

International audience; Epidemiologic studies have revealed that diets rich in sulforaphane (SFN), an isothiocyanate present in cruciferous vegetables, are associated with a marked decrease in prostate cancer incidence. The chemo-preventive role of SFN is associated with its histone de-acetylase inhibitor activity. However, the effect of SFN on chromatin composition and dynamic folding, especially in relation to HDAC inhibitor activity, remains poorly understood. In this study, we found that SFN can inhibit the expression and activity of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, in 2 prostate cancer cell lines. This decrease in gene expression is correlated with SFN-induced changes in chromatin structure and composition. The SFN-mediated changes in levels of histone post-translational modifications, more specifically acetylation of histone H3 lysine 18 and di-methylation of histone H3 lysine 4, 2 modifications linked with high risk of prostate cancer recurrence, were associated with regulatory elements within the hTERT promoter region. Chromatin condensation may also play a role in SFN-mediated hTERT repression, since expression and recruitment of MeCP2, a known chromatin compactor, were altered in SFN treated prostate cancer cells. Chromatin immuno-precipitation (ChIP) of MeCP2 showed enrichment over regions of the hTERT promoter with increased nucleosome density. These combined results strongly support a role for SFN in the mediation of epigenetic events leading to the repression of hTERT in prostate cancer cells. This ability of SFN to modify chromatin composition and structure associated with target gene expression provides a new model by which dietary phytochemicals may exert their chemoprevention activity.

Hereditary cancer syndromes serve as powerful models to uncover cancer-relevant genes. Cowden syndrome (CS), typically caused by germline PTEN mutations, is a heritable cancer syndrome with high risks of breast and thyroid cancers. However, there is a growing subset of CS without PTEN mutations. By exome sequencing cum family-studies, we identified heterozygous SEC23B variants in CS-associated and apparently sporadic thyroid cancer. Functional characterization revealed mutant CS-associated SEC23B led to endoplasmic reticulum (ER)-stress-mediated cell-colony formation and survival, growth and invasion, reflecting major hallmarks of cancer. Classically, SEC23B is a component of coat protein complex II (COPII) vesicles that transport secretory proteins from the ER to the Golgi apparatus. Germline homozygous or compound heterozygous loss-of-function SEC23B mutations cause Congenital Dyserythropoietic Anemia Type II (CDA II), resulting in decreased SEC23B levels. Intriguingly, our in vitro data does not recapitulate the CDA II-like decreased SEC23B protein expression, suggesting possible change-of-function effects in a cancer context. Here, we show mutant SEC23B exists within nucleoli, besides classical distribution at the ER/Golgi. This occurs in the absence of nucleolar localization of other COPII protein components and does not compromise canonical secretory function. Global transcriptomic and polysome profiling analyses reveal increased expression of ribosomal protein and translation-related genes, accompanied by enhanced translational capacity in mutant cells in response to ER stress. We also show mutant SEC23B directly binds to Upstream Binding Transcription Factor (UBF), concomitantly with increased UBF binding at the ribosomal DNA (rDNA) promoter region with ER stress. Unbiased proteomic analyses further show SEC23B, both wildtype and mutant, can interact with proteins in the ER stress and EIF2 signaling pathways. We validate through immunoprecipitation the binding interaction between SEC23B and UBA52, with notably increased binding with mutant SEC23B. UBA52 is ribosomal protein RPL40, known to regulate selective translation of stress-related transcripts, protein synthesis, and the cell cycle. Knockdown of SEC23B in vitro resulted in a concomitant decrease in protein levels of UBA52, further supporting that SEC23B and UBA52 are potentially co-regulated. All in all, our data suggest that SEC23B has non-canonical COPII-independent function, particularly within the ribosome biogenesis pathway, that may contribute to the pathogenesis of cancer-predisposition. Citation Format: Lamis Yehia, Ying Ni, Supriya Jindal, Ata Abbas, Anton A. Komar, Charis Eng. Noncanonical role of SEC23B in thyroid carcinogenesis: Anemia gene meets cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 400.

Epigenetic modifications play a key role in the patho-physiology of prostate cancer. Histone deacetylases (HDACs) play major roles in prostate cancer progression. HDACs are part of a transcriptional co-repressor complex that influences various tumor suppressor genes. Because of the significant roles played by HDACs in various human cancers, HDAC inhibitors are emerging as a new class of chemotherapeutic agents. HDAC inhibitors have been shown to induce cell growth arrest, differentiation and/or apoptosis in prostate cancer. The combined use of HDAC inhibitors with other chemotherapeutic agents or radiotherapy in cancer treatment has shown promising results. Various HDAC inhibitors are in different stages of clinical trials. In this review we discuss the molecular mechanism(s) through which HDACs influence prostate cancer progression, and the potential roles of HDAC inhibitors in prostate cancer prevention and therapy.

In neurocysticercosis, parasite-induced immune suppressive effects are thought to play an important role in enabling site-specific inhibition of inflammatory responses to infections. It is axiomatic that microglia-mediated (M1 proinflammatory) response causes central nervous system inflammation; however, the mechanisms by which helminth parasites modulate microglia activation remain poorly understood. Here, we show that microglia display a diminished expression of M1-inflammatory mediators such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and nitric oxide synthase 2 (NOS2) in murine neurocysticercosis. Microglia also exhibited a lack of myeloid cell maturation marker major histocompatibility complex (MHC)-II in these parasite-infected brains. Treatment of microglia with helminth soluble/secreted factors (HSFs) in vitro did not induce expression of M1-inflammatory signature molecule NOS2 as well as MHC-II in primary microglia. However, HSF treatment completely inhibited lipopolysaccharide-induced increase in expression of MHC-II, NOS2 and nitric oxide production in these cells. As epigenetic modulation of chromatin states that regulates recruitment of RNA polymerase II (Pol-II) is a key regulatory step in determining gene expression and functional outcome, we next evaluated whether HSF induced modulation of these phenomenon in microglia in vitro. Indeed, HSF downregulated Pol-II recruitment to the promoter region of TNF-α, IL-6, NOS2, MHC-II, and transcription factor CIITA (a regulator of MHC-II expression), by itself. Moreover, HSF suppressed the lipopolysaccharide-induced increase in Pol-II recruitment as well. In addition, HSF exposure reduced the positive histone marks H3K4Me3 and H3K9/14Ac at the promoter of TNF-α, IL-6, NOS2, MHC-II, and CIITA. These studies provide a novel mechanistic insight into helminth-mediated immune suppression in microglia via modulation of epigenetic processes.

Human leukocyte antigen (HLA)-G is a non-classical class I antigen. It has limited expression, but is high at the foetomaternal interface. This unique expression pattern of HLA-G suggests that it might be important for survival of the foetus during pregnancy. In the present study, 120 women with recurrent spontaneous abortions (RSA) and 120 fertile control women were genotyped for the HLA-G locus. This is the first report describing HLA-G polymorphism in normal fertile and RSA women from India. The allele HLA-G*010103 was higher in women with recurrent foetal losses. Interestingly, the HLA-G*010105 and G*010108 alleles were totally absent in normal fertile women but present in RSA women with frequencies of 1.7% and 0.4%, respectively. Allele G*010107 was absent in both the groups. The frequency of the null allele G*0105N was high (13.8%) in our population as compared to other world populations. Our data support the hypothesis that HLA-G polymorphism may contribute to recurrent foetal loss.