Angelman syndrome (AS) is a neurodevelopmental disorder in which epilepsy is common (~90%) and often refractory to antiepileptics. AS is caused by mutation of the maternal allele encoding the ubiquitin protein ligase E3A (UBE3A), but it is unclear how this genetic insult confers vulnerability to seizure development and progression (i.e., epileptogenesis). Here we implemented the flurothyl kindling and retest paradigm in AS model mice to assess epileptogenesis and to gain mechanistic insights owed to loss of maternal Ube3a. AS model mice kindled similarly to wildtype mice, but they displayed a markedly increased sensitivity to flurothyl-, kainic acid-, and hyperthermia-induced seizures measured a month later during retest. Pathological characterization revealed enhanced deposition of perineuronal nets in dentate gyrus of hippocampus of AS mice in the absence of overt neuronal loss or mossy fiber sprouting. This pro-epileptogenic phenotype resulted from Ube3a deletion in GABAergic but not glutamatergic neurons, and it was rescued by pancellular reinstatement of Ube3a at postnatal day 21 (P21), but not during adulthood. Our results suggest that epileptogenic susceptibility in AS patients is a consequence of the dysfunctional development of GABAergic circuits, which may be amenable to therapies leveraging juvenile reinstatement of UBE3A.
Bin Gu, Kelly E. Carstens, Matthew C. Judson, Katherine A. Dalton, Marie Rougié, Ellen P. Clark, Serena M. Dudek, Benjamin D. Philpot
Iron-related disorders are among the most prevalent diseases worldwide. Systemic iron homeostasis requires hepcidin, a hepatic-derived hormone that controls iron mobilization through its molecular target, ferroportin (FPN), the only known mammalian iron exporter. This pathway is perturbed in diseases that cause iron overload. Additionally, intestinal HIF-2α is essential for the local absorptive response to systemic iron deficiency and iron overload. Our data demonstrate a hetero-tissue crosstalk mechanism, where liver hepcidin regulated intestinal HIF-2α in iron deficiency, anemia, and iron overload. We show that FPN controlled cell autonomous iron efflux to regulate the activity of iron-dependent, intestinal prolyl hydroxylase domain enzymes to stabilize HIF-2α. Pharmacological blockade of HIF-2α using a clinically relevant and highly specific inhibitor successfully treated iron overload in a mouse model. These findings demonstrate a molecular link between liver hepcidin and intestinal HIF-2α that controls physiological iron uptake and drives iron hyperabsorption during iron overload.
Andrew J. Schwartz, Nupur K. Das, Sadeesh K. Ramakrishnan, Chesta Jain, Mladen Jurkovic, Jun Wu, Elizabeta Nemeth, Samira Lakhal-Littleton, Justin A. Colacino, Yatrik M. Shah
The loss of insulin-secreting β cells is characteristic among Type I and Type II diabetes. Stimulating proliferation to expand sources of β cells for transplantation remains a challenge because adult β cells do not proliferate readily. The cell cycle inhibitor p57 has been shown to control cell division in human β cells. Expression of p57 is regulated by the DNA methylation status of the Imprinting Control Region 2 (ICR2), which is commonly hypomethylated in Beckwith Wiedemann-Syndrome patients who exhibit massive β cell proliferation. We hypothesized that targeted demethylation of the ICR2 using a transcription activator-like effector protein fused to the catalytic domain of TET1 (ICR2-TET1) would repress p57 expression and promote cell proliferation. We report here that overexpression of ICR2-TET1 in human fibroblasts reduces p57 expression levels and increases proliferation. Furthermore, human islets overexpressing ICR2-TET1 exhibit repression of p57 with concomitant upregulation of Ki-67 while maintaining glucose-sensing functionality. When transplanted into diabetic, immunodeficient mice, the epigenetically edited islets show increased β cell replication compared to control islets. These findings demonstrate that epigenetic editing is a promising tool for inducing β cell proliferation, which may one day alleviate the scarcity of transplantable β cells for the treatment of diabetes.
Kristy Ou, Ming Yu, Nicholas G. Moss, Yue J. Wang, Amber W. Wang, Son C. Nguyen, Connie Jiang, Eseye Feleke, Vasumathi Kameswaran, Eric F. Joyce, Ali Naji, Benjamin Glaser, Dana Avrahami, Klaus H. Kaestner
BACKGROUND. Liquid biopsies have demonstrated that the constitutively active androgen receptor splice variant-7 (AR-V7) associates with reduced response and overall survival (OS) from endocrine therapies in castration resistant prostate cancer (CRPC). However, these studies provide little information pertaining to AR-V7 expression in prostate cancer (PC) tissue. METHODS. Following generation and validation of a novel AR-V7 antibody for immunohistochemistry, AR-V7 protein expression was determined for 358 primary prostate samples and 293 metastatic biopsies. Associations with disease progression, full length AR (AR-FL) expression, response to therapy, and gene expression was determined. RESULTS. We demonstrated that AR-V7 protein is rarely expressed (<1%) in primary PC but is frequently detected (75% of cases) following androgen deprivation therapy, with further significant (P = 0.020) increase in expression following abiraterone acetate or enzalutamide therapy. In CRPC, AR-V7 expression is predominantly (94% of cases) nuclear and correlates with AR-FL expression (P ≤ 0.001) and AR copy number (P = 0.026). However, dissociation of expression was observed suggesting mRNA splicing remains crucial for AR-V7 generation. AR-V7 expression was heterogeneous between different metastases from a patient although AR-V7 expression was similar within a metastasis. Moreover, AR-V7 expression correlated with a unique 59-gene signature in CRPC, including HOXB13, a critical co-regulator of AR-V7 function. Finally, AR-V7 negative disease associated with better PSA responses (100% vs 54%; P = 0.03) and OS (74.3 vs 25.2mo, HR 0.23 [0.07-0.79], P = 0.02) from endocrine therapies (pre-chemotherapy). CONCLUSION. This study provides impetus to develop therapies that abrogate AR-V7 signaling to improve our understanding of AR-V7 biology, and to confirm its clinical significance.
Adam Sharp, Ilsa Coleman, Wei Yuan, Cynthia Sprenger, David Dolling, Daniel Nava Rodrigues, Joshua W. Russo, Ines Figueiredo, Claudia Bertan, George Seed, Ruth Riisnaes, Takuma Uo, Antje Neeb, Jonathan Welti, Colm Morrissey, Suzanne Carreira, Jun Luo, Peter S. Nelson, Steven P. Balk, Lawrence D. True, Johann De Bono, Stephen R. Plymate
The pathogenesis of ischemic diseases remains unclear. Here we demonstrate the induction of microRNA-668 (mir-668) in ischemic acute kidney injury (AKI) in human patients, mice, and renal tubular cells. The induction was HIF-1dependant as HIF-1-deficiency in cells and kidney proximal tubules attenuated mir-668 expression. We further identified a functional HIF-1 binding site in mir-668 gene promoter. Anti-mir-668 increased apoptosis in renal tubular cells and enhanced ischemic AKI in mice, whereas mir-668 mimic was protective. Mechanistically, anti-mir-668 induced mitochondrial fragmentation, whereas mir-668 blocked mitochondrial fragmentation during hypoxia. We analyzed mir-668 target genes through immunoprecipitation of microRNA-induced silencing complexes followed by RNA deep sequencing and identified 124 protein-coding genes as likely targets of mir-668. Among these genes, only Mitochondrial Protein 18 KDa (MTP18) has been implicated in mitochondrial dynamics. In renal cells and mouse kidneys, mir-668 mimic suppressed MTP18, whereas anti-mir-668 increased MTP18 expression. Luciferase microRNA target reporter assay further verified MTP18 as a direct target of mir-668. In renal tubular cells, knockdown of MTP18 suppressed mitochondrial fragmentation and apoptosis. Together, the results suggest that mir-668 is induced via HIF-1 in ischemic AKI and, upon induction, mir-668 represses MTP18 to preserve mitochondrial dynamics for renal tubular cell survival and kidney protection.
Qingqing Wei, Haipeng Sun, Shuwei Song, Yong Liu, Pengyuan Liu, Man J. Livingston, Jianwen Wang, Mingyu Liang, Qing-Sheng Mi, Yuqing Huo, N. Stanley Nahman, Changlin Mei, Zheng Dong
Mutations in CDCA7 and HELLS that respectively encode a CXXC-type zinc finger protein and a SNF2 family chromatin remodeler cause immunodeficiency, centromeric instability, facial anomalies (ICF) syndrome type 3 and 4, respectively. Here, we demonstrate that classical non-homologous end joining (C-NHEJ) proteins Ku80 and Ku70, as well as HELLS coimmunoprecipitated with CDCA7. The coimmunoprecipitation of the repair proteins was sensitive to nuclease treatment and an ICF3 mutation in CDCA7 that impairs its chromatin binding. The functional importance of these interactions was strongly suggested by the compromised C-NHEJ activity and significant delay in Ku80 accumulation at DNA damage sites in CDCA7 and HELLS deficient HEK293 cells. Consistent with the repair defect, these cells displayed increased apoptosis, abnormal chromosome segregation, aneuploidy, centrosome amplification, and significant accumulation of γH2AX signals. Although less prominent, cells mutated for the other ICF genes DNMT3B and ZBTB24 (responsible for ICF type 1 and 2, respectively) showed similar defects. Importantly, lymphoblastoid cells from ICF patients shared the same changes detected in the mutant HEK293 cells to varying degrees. Although the C-NHEJ defect alone did not cause CG hypomethylation, CDCA7 and HELLS are involved in maintaining CG methylation at centromeric and pericentromeric repeats. The defect in C-NHEJ may account for some common features of ICF cells, including centromeric instability, abnormal chromosome segregation, and apoptosis.
Motoko Unoki, Hironori Funabiki, Guillaume Velasco, Claire Francastel, Hiroyuki Sasaki
Glioblastoma is highly enriched with macrophages, and osteopontin (OPN) expression levels correlate with glioma grade and the degree of macrophage infiltration, thus we studied whether OPN plays a crucial role in immune modulation. Quantitative PCR, immune blotting, and ELISA were used to determine OPN expression. Knockdown of OPN was achieved using complementary siRNA, shRNA and CRISPR/CAS9 techniques followed by a series of in vitro functional migration and immunological assays. OPN gene-deficient mice were used to examine the roles of non-tumor-derived OPN on survival of mice harboring intracranial gliomas. Patients with mesenchymal GBM show high OPN expression, a negative survival prognosticator. OPN is a potent chemokine for macrophages, and its blockade significantly impaired the ability of glioma cells to recruit macrophages. Integrin αVβ5 (ITGαVβ5) is highly expressed on glioblastoma-infiltrating macrophages and constitutes a major OPN receptor. OPN maintains the M2 macrophage gene signature and phenotype. Both tumor-derived OPN and host-derived OPN was critical for glioma development. OPN deficiency in either the innate immune or glioma cells demonstrated a marked reduction of M2 macrophages and elevated T cell effector activity infiltrating the glioma. Furthermore, OPN deficiency in the glioma cells sensitized them to direct CD8+ T cell cytotoxicity. OPN can be exploited as an immune modulatory target, with efficacious therapeutic results using systemically administered OPN-4-1BB bispecific aptamers, increasing median survival time by 68% (P < 0.05). OPN is an important chemokine for recruiting macrophages to glioblastoma, mediates crosstalk between tumor cells and the innate immune system, and can be exploited as a therapeutic target.
Jun Wei, Anantha Marisetty, Brett Schrand, Konrad Gabrusiewicz, Yuuri Hashimoto, Martina Ott, Zacharia Grami, Ling-Yuan Kong, Xiaoyang Ling, Hillary G. Caruso, Shouhao Zhou, Y. Alan Wang, Gregory N. Fuller, Jason T. Huse, Eli Gilboa, Nannan Kang, Xingxu Huang, Roel Verhaak, Shulin Li, Amy B. Heimberger
Transplantation with autologous hematopoietic progenitors remains an important consolidation treatment for multiple myeloma (MM) patients and is thought to prolong disease plateau-phase by providing intensive cytoreduction. However, transplantation induces inflammation in the context of profound lymphodepletion that may cause hitherto unexpected immunological effects. We developed preclinical models of bone marrow transplantation (BMT) for MM using Vk*MYC myeloma-bearing recipients and donors that were myeloma-naïve or were myeloma-experienced to simulate autologous transplantation. Surprisingly, we demonstrate broad induction of T cell-dependent myeloma control, most efficiently from memory T cells within myeloma-experienced grafts, but also through priming of naïve T cells after BMT. CD8+ T cells from mice with controlled myeloma had a distinct TCR repertoire and higher clonotype overlap relative to myeloma-free BMT recipients. Furthermore, T cell-dependent myeloma control could be adoptively transferred to secondary recipients, and was myeloma clone-specific. Interestingly, donor-derived IL-17A acted directly on myeloma cells expressing the IL-17-receptor to induce a transcriptional landscape that promoted tumor growth and immune escape. Conversely, donor IFNγ secretion and signaling was critical to protective immunity, and was profoundly augmented by CD137 agonists. These data provide new insights into the mechanisms of action of transplantation in myeloma and suggests rational approaches to improving clinical outcome.
Slavica Vuckovic, Simone A. Minnie, David Smith, Kate H. Gartlan, Thomas S. Watkins, Kate A. Markey, Pamela Mukhopadhyay, Camille Guillerey, Rachel D. Kuns, Kelly R. Locke, Antonia L. Pritchard, Peter A. Johansson, Antiopi Varelias, Ping Zhang, Nicholas D. Huntington, Nicola Waddell, Marta Chesi, John J. Miles, Mark J. Smyth, Geoffrey R. Hill
Activating mutations in the Wnt pathway drive a variety of cancers, but the specific targets and pathways activated by Wnt ligands are not fully understood. To bridge this knowledge gap, we performed a comprehensive time-course analysis of Wnt-dependent signaling pathways in an orthotopic model of Wnt-addicted pancreatic cancer, using a PORCN inhibitor currently in clinical trials, and validated key results in additional Wnt-addicted models. The temporal analysis of the drug-perturbed transcriptome demonstrated direct and indirect regulation of greater than 3,500 Wnt activated genes (23% of the transcriptome). Regulation was both via Wnt/β-catenin, and through the modulation of protein abundance of important transcription factors including MYC via Wnt/STOP. Our study identifies a central role of Wnt /β-catenin and Wnt/STOP signaling in controlling ribosomal biogenesis, a key driver of cancer proliferation.
Babita Madan, Nathan Harmston, Gahyathiri Nallan, Alex Montoya, Peter Faull, Enrico Petretto, David M. Virshup
Non-alcoholic fatty liver disease (NAFLD) arises from mitochondrial dysfunction under sustained imbalance between energy intake and expenditure, but the underlying mechanisms controlling mitochondrial respiration have not been entirely understood. Heterotrimeric G proteins converge signals from activated GPCRs, and modulate cell signaling pathways to maintain metabolic homeostasis. Here, we investigated the regulatory role of Gα12 on hepatic lipid metabolism and whole-body energy expenditure in mice. Fasting increased Gα12 level in mouse liver. Gα12 ablation markedly augmented fasting-induced hepatic fat accumulation. cDNA microarray analysis from Gna12 KO liver revealed that Gα12 signaling pathway regulated sirtuin 1 (SIRT1) and PPARα responsible for mitochondrial respiration. Defective induction of SIRT1 upon fasting was observed in the liver of Gna12 KO mice, which was reversed by lentivirus-mediated Gα12 overexpression in hepatocytes. Mechanistically, Gα12 stabilized SIRT1 protein through transcriptional induction of USP22 via HIF-1α increase. Gα12 levels were markedly diminished in liver biopsies from NAFLD patients. Consistently, Gna12 KO mice fed high-fat diet displayed greater susceptibility to diet-induced liver steatosis and obesity due to decrease in energy expenditure. Our results demonstrate that Gα12 regulates SIRT1-dependent mitochondrial respiration through HIF-1α-dependent USP22 induction, identifying Gα12 as an upstream molecule that contributes to the regulation of mitochondrial energy expenditure.
Tae Hyun Kim, Yoon Mee Yang, Chang Yeob Han, Ja Hyun Koo, Hyunhee Oh, Su Sung Kim, Byoung Hoon You, Young Hee Choi, Tae-Sik Park, Chang Ho Lee, Hitoshi Kurose, Mazen Noureddin, Ekihiro Seki, Yu-Jui Yvonne Wan, Cheol Soo Choi, Sang Geon Kim
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