Citations

Abstract The amplified MYCN gene serves as an oncogenic driver in approximately 20% of high-risk pediatric neuroblastomas. Here we show that c-MYC itself is a potent transforming gene in a separate subset of high-risk neuroblastoma cases (~10%), based on (i) its upregulation by focal enhancer amplification or genomic rearrangements leading to enhancer hijacking, and (ii) its ability to transform neuroblastoma precursor cells in a transgenic animal model. The aberrant regulatory elements associated with oncogenic c-MYC activation include focally amplified distal enhancers or translocation of highly active enhancers from other genes to within topologically associating domains containing the c-MYC gene locus. The clinical outcome for patients with high levels of c-MYC expression is virtually identical to that of patients with amplification of the MYCN gene, a known high-risk feature of this disease. Together, these findings establish c-MYC as a bona fide oncogene in a clinically significant group of high risk childhood neuroblastomas. Citation Format: Mark W. Zimmerman, Yu Liu, Shuning He, Adam D. Durbin, Brian J. Abraham, John Easton, Ying Shao, Xu Beisi, Shizhen Zhu, Xiaoling Zhang, Zhaodong Li, Nina Weichert-Leahey, Richard A. Young, Jinghui Zhang, A. Thomas Look. c-MYC drives a subset of high-risk pediatric neuroblastoma and is activated through mechanisms including enhancer hijacking and focal enhancer amplification [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 4135.

Abstract When unrepaired, alkylated DNA can induce cell death or trigger mutagenesis. Cellular capacity for repair of these lesions by O6-methylguanine methyltransferase (MGMT) dictates the equilibrium between cell viability and genetic diversity. Treatment of glioblastoma cells with temozolomide (TMZ) induced ATM- and Rad3-related (ATR) kinase dependent polyribonucleotide nucleotidyltransferase 1 (PNPT1) degradation of miR-181d. miR-181d suppresses MGMT expression; its degradation increases the mean MGMT expression of the cell population. miR-181d degradation also magnifies the cell-to-cell variability in MGMT expression, expanding the genetic heterogeneity of the population. This expanded heterogeneity enhances the “fitness” of the population and constitutes a novel form of chemotherapeutic resistance. These effects can be suppressed by overexpression of miR-181d, suggesting miRNA delivery as a strategy for glioblastoma therapy. To characterize the mechanism of acquired resistance, we profiled the expression of 2400 miRNAs before and after TMZ treatment. In independent patient-derived neurosphere lines, the majority of miRNAs remained unchanged after treatment. However, miR-181d was consistently suppressed after TMZ treatment. Our previous work demonstrated miR-181d as the master regulator of MGMT. We confirmed TMZ-induced suppression of miR-181d using independent in vitro and in vivo models as well as matched pre- and post-TMZ treated clinical specimens. TMZ-induced miR-181d suppression persisted after transcriptional inhibition, suggesting degradation as the primary mechanism. We performed an siRNA screen and identified polyribonucleotide nucleotidyltransferase 1 (PNPT1) as the gene responsible for miR-181d degradation. CRISPR inactivation of PNPT1 eliminated TMZ-induced suppression of miR-181d; this was rescued by wild-type PNPT1 but not by PNPT1 harboring RNAse-inactivating mutations. TMZ-induced degradation of miR-181d requires ATR kinase. Silencing or inhibition of ATR eliminated binding of PNPT1 to miR-181d and prevented degradation of miR-181d. TMZ-sensitizing effects of ATR inhibition were reversed by anti-miR-181d, suggesting miR-181d is essential in this process. In addition to elevating the mean MGMT expression of the population, single-cell analysis revealed that miR-181d degradation broadened the cell-to-cell variability in MGMT expression in vitro. In matched clinical pre- and post-TMZ treated specimens, variability in MGMT expression was significantly elevated in post-TMZ samples. This was recapitulated using The Cancer Genome Atlas (TCGA) database. We propose that miR-181d degradation-mediated expansion of genetic heterogeneity enhances the “fitness” of the population, constituting a novel form of chemotherapeutic resistance. These effects are suppressed by miR-181d overexpression, suggesting miRNA delivery as a strategy for glioblastoma therapy. Citation Format: Valya Ramakrishnan, Johnny Akers, Thien Nguyen, Aaron Wang, Bandita Adhikari, Brian Hirshman, Jie Li, Jann Sarkaria, Wei Hua, Mao Ying, Masayuki Nitta, Tao Jiang, Bob Carter, Clark C. Chen. miR-181d degradation mediated genetic heterogeneity and acquired resistance [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 1956.

ABSTRACT Dimethylarginine dimethylaminohydrolases (DDAHs) are known to degrade asymmetric dimethylarginine, an endogenous inhibitor of NOS, and maintain vascular homeostasis; however, the regulatory pathways of DDAHs remain unclear. In this study, we aimed to define the role of transmembrane glycoprotein neuropilin‐1 (NRP1) in the expression of DDAHs and investigate the potential roles of NRP1 in regulation of blood pressure. Short hairpin RNA–mediated knockdown of NRP1 reduced the level and mRNA stability of DDAH1 but not DDAH2 in HUVECs, whereas overexpression of NRP1 increased the mRNA stability of DDAH1. Meanwhile, mesenteric arteries and lung vascular endothelial cells of tamoxifen‐inducible endothelial cell–specific NRP1 knockout mice exhibited decreased expression of DDAH1 and slightly increased expression of DDAH2. Mechanistically, the regulation of NRP1 on DDAH1 expression is mediated by a posttranscriptional mechanism involving miR‐219‐5p in HUVECs. Although the endothelial cell–specific NRP1 knockout mice did not exhibit any significant change in blood pressure at the basal level, they were more sensitive to low‐dose angiotensin II infusion–induced increases in blood pressure. Our results show that NRP1 is required for full expression of DDAH1 in endothelial cells and that NRP1 contributes to protection from low‐dose angiotensin II–induced increases in blood pressure.—Wang, Y., Wang, E., Zhang, Y., Madamsetty, V. S., Ji, B., Radisky, D. C., Grande, J. P., Misra, S., Mukhopadhyay, D. Neuropilin‐1 maintains dimethylarginine dimethylaminohydrolase 1 expression in endothelial cells, and contributes to protection from angiotensin II–induced hypertension. FASEB J. 33, 494–500 (2019). www.fasebj.org

ObjectivePrevious studies suggest that the brain‐derived neurotrophic factor (BDNF) Val66Met (rs6265) polymorphism may influence symptom onset in Alzheimer's disease (AD). Our recent cross‐sectional findings suggest that Met66 may influence clinical expression in dominantly inherited AD (DIAD) through its effects on tau. However, it remains unclear whether carriage of Met66 in DIAD results in faster increases in cerebrospinal fluid (CSF) tau and ptau181, and whether these increases are associated with accelerated brain volume loss and memory decline.MethodsA total of 211 subjects (101 mutation noncarriers, 110 mutation carriers), who were cognitively normal, as defined by a Clinical Dementia Rating global score of 0, completed assessments of cognitive function, neuroimaging, and CSF sampling over 3.5 years as part of the Dominantly Inherited Alzheimer's Network.ResultsIn mutation carriers, Met66 carriers showed faster memory decline (4×), hippocampal volume loss (16×), and CSF tau and ptau181increases (6×) than Val66 homozygotes.BDNFdid not influence rates of cortical β‐amyloid accumulation or change in CSF Aβ42levels in mutation carriers. In mutation noncarriers,BDNFgenotype had no effect on change in cognition, brain volume, cortical β‐amyloid accumulation, or change in any CSF measures of tau, ptau181, and CSF Aβ42.InterpretationAs in sporadic AD, the deleterious effects of β‐amyloid on cognitive function, brain volume loss, and CSF tau in DIAD mutation carriers are less in Val66 homozygotes. TheBDNFVal66Met polymorphism should be considered as a potential moderator of clinical trial outcomes in current treatment and prevention trials in DIAD and sporadic AD. Ann Neurol 2018;84:424–435

The mechanisms underlying proangiogenic function of brain-derived neurotrophic factor (BDNF) are not fully understood. The current study was designed to explore the microRNA (miRNA) profile in human early endothelial progenitor cells (EPCs, also referred to as CFU-Hill cells) treated with BDNF. Treatment of early EPCs with BDNF for 7 d significantly increased the colony formation of outgrowth endothelial cells. BDNF suppressed the expression of miR-4716-5p, miR-3928, miR-433, miR-1294, miR-1539, and miR-19b-1*. In contrast, BDNF significantly increased the levels of miR-432*, miR-4499, miR-3911, miR-1183, miR-4669, miR-636, miR-4717-3p, miR-4298, miR485-5p, and miR-181c. Since miR-433 has been reported to augment hematopoietic cells proliferation and differentiation, we examined the role of miR-433 in regenerative effects of BDNF. BDNF stimulated the protein expression of guanylate-binding protein 2 via the suppression of miR-433. However, the knockdown of miR-433 was not sufficient to significantly increase the number of outgrowth endothelial cell colonies, suggesting that modulation of miR-433 alone does not stimulate regenerative capacity of EPCs. In aggregate, our results also suggest that the effect of BDNF on regenerative function of EPCs may depend on complex changes in the expression of microRNAs.

Although immune checkpoint inhibitors have resulted in durable clinical benefits in a subset of patients with advanced cancer, some patients who did not respond to initial anti–PD-1 therapy have been found to benefit from the addition of salvage chemotherapy. However, the mechanism responsible for the successful chemoimmunotherapy is not completely understood. Here we show that a subset of circulating CD8 + T cells expressing the chemokine receptor CX3CR1 are able to withstand the toxicity of chemotherapy and are increased in patients with metastatic melanoma who responded to chemoimmunotherapy (paclitaxel and carboplatin plus PD-1 blockade). These CX3CR1 + CD8 + T cells have effector memory phenotypes and the ability to efflux chemotherapy drugs via the ABCB1 transporter. In line with clinical observation, our preclinical models identified an optimal sequencing of chemoimmunotherapy that resulted in an increase of CX3CR1 + CD8 + T cells. Taken together, we found a subset of PD-1 therapy–responsive CD8 + T cells that were capable of withstanding chemotherapy and executing tumor rejection with their unique abilities of drug efflux (ABCB1), cytolytic activity (granzyme B and perforin), and migration to and retention (CX3CR1 and CD11a) at tumor sites. Future strategies to monitor and increase the frequency of CX3CR1 + CD8 + T cells may help to design effective chemoimmunotherapy to overcome cancer resistance to immune checkpoint blockade therapy.

Abstract Purpose: Homozygous deletions play important roles in carcinogenesis. The genome-wide screening for homozygously deleted genes in many different cancer types with a large number of patient specimens representing the tumor heterogeneity has not been done. Experimental Design: We performed integrative analyses of the copy-number profiles of 10,759 patients across 31 cancer types from The Cancer Genome Atlas project. Results: We found that the type-I interferon, α-, and β-defensin genes were homozygously deleted in 19 cancer types with high frequencies (7%–31%, median = 12%; interquartile range = 10%–16.5%). Patients with homozygous deletion of interferons exhibited significantly shortened overall or disease-free survival time in a number of cancer types, whereas patients with homozygous deletion of defensins did not significantly associate with worse overall or disease-free survival. Gene expression analyses suggested that homozygous deletion of interferon and defensin genes could activate genes involved in oncogenic and cell-cycle pathways but repress other genes involved in immune response pathways, suggesting their roles in promoting tumorigenesis and helping cancer cells evade immune surveillance. Further analysis of the whole exomes of 109 patients with melanoma demonstrated that the homozygous deletion of interferon (P = 0.0029, OR = 11.8) and defensin (P = 0.06, OR = 2.79) genes are significantly associated with resistance to anti-CTLA4 immunotherapy. Conclusions: Our analysis reveals that the homozygous deletion of interferon and defensin genes is prevalent in human cancers, and importantly this feature can be used as a novel prognostic biomarker for immunotherapy resistance. Clin Cancer Res; 24(14); 3299–308. ©2018 AACR.

Mitochondria are dynamic organelles that are able to change their morphology and cellular distribution by either fission or fusion. However, the molecular mechanisms controlling mitochondrial dynamics in vascular endothelial cells (ECs) remain largely unknown. In this study, we observed that knockdown of microtubule‐associated tumor suppressor 1 (MTUS1) in ECs inhibited tube formation and migration, accompanied with decreased promigratory signalings. We showed that MTUS1 was localized in the outer membrane of mitochondria in ECs. Knockdown of MTUS1 disturbed the elongated mitochondrial network and induced the formation of perinuclear clusters of mitochondria. Importantly, mitochondrial motility and fusion were suppressed, whereas generation of reactive oxygen species was increased in MTUS1 knockdown ECs. Mechanistically, we showed that the N‐terminal coiled‐coil domain of MTUS1 interacted with the mitochondrial membrane proteins, mitofusin‐1 and mitofusin‐2, to maintain mitochondrial morphology in ECs. This study illustrated a novel role of MTUS1 in mitochondrial morphology and EC angiogenic responses.—Wang Y., Huang Y., Liu, Y., Li, J., Hao, Y., Yin, P., Liu, Z., Chen J., Wang Y., Wang N., Zhang P., Microtubule associated tumor suppressor 1 interacts with mitofusins to regulate mitochondrial morphology in endothelial cells. FASEB J . 32, 4504–4518 (2018). www.fasebj.org