Genetic Phenomena Commons^™

Immune Landscape Of A Genetically Engineered Murine Model Of Glioma Compared With Human Glioma, Daniel B Zamler, Takashi Shingu, Laura M Kahn, Kristin Huntoon, Cynthia Kassab, Martina Ott, Katarzyna Tomczak, Jintan Liu, Yating Li, Ivy Lai, Rocio Zorilla-Veloz, Cassian Yee, Kunal Rai, Betty Ys Kim, Stephanie S Watowich, Amy B Heimberger, Giulio F Draetta, Jian Hu

Faculty, Staff and Student Publications

Novel therapeutic strategies targeting glioblastoma (GBM) often fail in the clinic, partly because preclinical models in which hypotheses are being tested do not recapitulate human disease. To address this challenge, we took advantage of our previously developed spontaneous Qk/Trp53/Pten (QPP) triple-knockout model of human GBM, comparing the immune microenvironment of QPP mice with that of patient-derived tumors to determine whether this model provides opportunity for gaining insights into tumor physiopathology and preclinical evaluation of therapeutic agents. Immune profiling analyses and single-cell sequencing of implanted and spontaneous tumors from QPP mice and from patients with glioma revealed intratumoral immune components that …

Integrated Screens Uncover A Cell Surface Tumor Suppressor Gene Kirrel Involved In Hippo Pathway, Chao Wang, Xu Feng, Dan Su, Zhen Chen, Shimin Wang, Mengfan Tang, Min Huang, Litong Nie, Huimin Zhang, Siting Li, Ling Yin, Randy L Johnson, Traver Hart, Junjie Chen

Faculty, Staff and Student Publications

Cell surface proteins play essential roles in various biological processes and are highly related to cancer development. They also serve as important markers for cell identity and targets for pharmacological intervention. Despite their great potentials in biomedical research, comprehensive functional analysis of cell surface proteins remains scarce. Here, with a de novo designed library targeting cell surface proteins, we performed in vivo CRISPR screens to evaluate the effects of cell surface proteins on tumor survival and proliferation. We found that

Directed Evolution Of Pd-L1-Targeted Affibodies By Mrna Display, Brian J Grindel, Brian J Engel, Justin N Ong, Anupallavi Srinivasamani, Xiaowen Liang, Niki M Zacharias, Robert C Bast, Michael A Curran, Terry T Takahashi, Richard W Roberts, Steven W Millward

Faculty, Staff and Student Publications

Therapeutic monoclonal antibodies directed against PD-L1 (e.g., atezolizumab) disrupt PD-L1:PD-1 signaling and reactivate exhausted cytotoxic T-cells in the tumor compartment. Although anti-PD-L1 antibodies are successful as immune checkpoint inhibitor (ICI) therapeutics, there is still a pressing need to develop high-affinity, low-molecular-weight ligands for molecular imaging and diagnostic applications. Affibodies are small polypeptides (∼60 amino acids) that provide a stable molecular scaffold from which to evolve high-affinity ligands. Despite its proven utility in the development of imaging probes, this scaffold has never been optimized for use in mRNA display, a powerful

Chemotherapy Coupled To Macrophage Inhibition Induces T-Cell And B-Cell Infiltration And Durable Regression In Triple-Negative Breast Cancer, Swarnima Singh, Nigel Lee, Diego A Pedroza, Igor L Bado, Clark Hamor, Licheng Zhang, Sergio Aguirre, Jingyuan Hu, Yichao Shen, Yitian Xu, Yang Gao, Na Zhao, Shu-Hsia Chen, Ying-Wooi Wan, Zhandong Liu, Jeffrey T Chang, Daniel Hollern, Charles M Perou, Xiang H F Zhang, Jeffrey M Rosen

Duncan NRI Faculty and Staff Publications

Immunosuppressive elements within the tumor microenvironment, such as tumor-associated macrophages (TAM), can present a barrier to successful anti-tumor responses by cytolytic T cells. Here we employed preclinical syngeneic p53 null mouse models of triple-negative breast cancer (TNBC) to develop a treatment regimen that harnessed the immunostimulatory effects of low-dose cyclophosphamide coupled with the pharmacologic inhibition of TAMs using either a small molecule CSF1R inhibitor or an anti-CSF1R antibody. This therapeutic combination was effective in treating several highly aggressive TNBC murine mammary tumor and lung metastasis models. Single cell RNA sequencing characterized tumor-infiltrating lymphocytes (TIL) including helper T cells and antigen-presenting …

Genome-Wide Crispr Screens Using Isogenic Cells Reveal Vulnerabilities Conferred By Loss Of Tumor Suppressors, Rodney Cheng-En Hsieh, Sunil Krishnan, Ren-Chin Wu, Akash R Boda, Arthur Liu, Michelle Winkler, Wen-Hao Hsu, Steven Hsesheng Lin, Mien-Chie Hung, Li-Chuan Chan, Krithikaa Rajkumar Bhanu, Anupallavi Srinivasamani, Ricardo Alexandre De Azevedo, Yung-Chih Chou, Ronald A Depinho, Matthew Gubin, Eduardo Vilar, Chao Hsien Chen, Ravaen Slay, Priyamvada Jayaprakash, Shweta Mahendra Hegde, Genevieve Hartley, Spencer T Lea, Rishika Prasad, Brittany Morrow, Coline Agnes Couillault, Madeline Steiner, Chun-Chieh Wang, Bhanu Prasad Venkatesulu, Cullen Taniguchi, Yon Son Betty Kim, Junjie Chen, Nils-Petter Rudqvist, Michael A Curran

Faculty, Staff and Student Publications

Radiotherapy (RT) of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)-expressing CRC cells systemically. However, abscopal tumor remissions are extremely rare, and the postirradiation immune escape mechanisms in CRC remain elusive. Here, we found that irradiated CRC cells used ATR-mediated DNA repair signaling pathway to up-regulate both CD47 and PD-L1, which through engagement of SIRPα and PD-1, respectively, prevented phagocytosis by antigen-presenting cells and thereby limited TAA cross-presentation and innate immune activation. This postirradiation CD47 and PD-L1 up-regulation was observed across various human solid tumor cells. Concordantly, rectal cancer patients with poor responses to neoadjuvant RT exhibited …

Atr-Mediated Cd47 And Pd-L1 Up-Regulation Restricts Radiotherapy-Induced Immune Prming And Abscopal Responses In Colorectal Cancer, Rodney Cheng-En Hsieh, Sunil Krishnan, Ren-Chin Wu, Akash R Boda, Arthur Liu, Michelle Winkler, Wen-Hao Hsu, Steven Hsesheng Lin, Mien-Chie Hung, Li-Chuan Chan, Krithikaa Rajkumar Bhanu, Anupallavi Srinivasamani, Ricardo Alexandre De Azevedo, Yung-Chih Chou, Ronald A Depinho, Matthew Gubin, Eduardo Vilar, Chao Hsien Chen, Ravaen Slay, Priyamvada Jayaprakash, Shweta Mahendra Hegde, Genevieve Hartley, Spencer T Lea, Rishika Prasad, Brittany Morrow, Coline Agnes Couillault, Madeline Steiner, Chun-Chieh Wang, Bhanu Prasad Venkatesulu, Cullen Taniguchi, Yon Son Betty Kim, Junjie Chen, Nils-Petter Rudqvist, Michael A Curran

Faculty, Staff and Student Publications

Radiotherapy (RT) of colorectal cancer (CRC) can prime adaptive immunity against tumor-associated antigen (TAA)-expressing CRC cells systemically. However, abscopal tumor remissions are extremely rare, and the postirradiation immune escape mechanisms in CRC remain elusive. Here, we found that irradiated CRC cells used ATR-mediated DNA repair signaling pathway to up-regulate both CD47 and PD-L1, which through engagement of SIRPα and PD-1, respectively, prevented phagocytosis by antigen-presenting cells and thereby limited TAA cross-presentation and innate immune activation. This postirradiation CD47 and PD-L1 up-regulation was observed across various human solid tumor cells. Concordantly, rectal cancer patients with poor responses to neoadjuvant RT exhibited …

Targeting Il-1Β As An Immunopreventive And Therapeutic Modality For K-Ras-Mutant Lung Cancer, Bo Yuan, Michael J Clowers, Walter V Velasco, Stephen Peng, Qian Peng, Yewen Shi, Marco Ramos-Castaneda, Melody Zarghooni, Shuanying Yang, Rachel L Babcock, Seon Hee Chang, John V Heymach, Jianjun Zhang, Edwin J Ostrin, Stephanie S Watowich, Humam Kadara, Seyed Javad Moghaddam

Faculty, Staff and Student Publications

K-ras-mutant lung adenocarcinoma (KM-LUAD) is associated with abysmal prognosis and is tightly linked to tumor-promoting inflammation. A human mAb, canakinumab, targeting the proinflammatory cytokine IL-1β, significantly decreased the risk of lung cancer in the Canakinumab Anti-inflammatory Thrombosis Outcomes Study. Interestingly, we found high levels of IL-1β in the lungs of mice with K-rasG12D-mutant tumors (CC-LR mice). Here, we blocked IL-1β using an anti-IL-1β mAb in cohorts of 6- or 14-week-old CC-LR mice to explore its preventive and therapeutic effect, respectively. IL-1β blockade significantly reduced lung tumor burden, which was associated with reprogramming of the lung microenvironment toward an antitumor phenotype …

Phosphorylation And Stabilization Of Pd-L1 By Ck2 Suppresses Dendritic Cell Function, Xixi Zhao, Yongkun Wei, Yu-Yi Chu, Yintao Li, Jung-Mao Hsu, Zhou Jiang, Chunxiao Liu, Jennifer L Hsu, Wei-Chao Chang, Riyao Yang, Li-Chuan Chan, Jingkun Qu, Shuqun Zhang, Haoqiang Ying, Dihua Yu, Mien-Chie Hung

Faculty, Staff and Student Publications

UNLABELLED: Targeting immune checkpoints such as programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) has transformed cancer treatment, with durable clinical responses across a wide range of tumor types. However, a high percentage of patients fail to respond to anti-PD-1/PD-L1 treatment. A greater understanding of PD-L1 regulation is critical to improving the clinical response rate of PD-1/PD-L1 blockade. Here, we demonstrate that PD-L1 is phosphorylated and stabilized by casein kinase 2 (CK2) in cancer and dendritic cells (DC). Phosphorylation of PD-L1 at Thr285 and Thr290 by CK2 disrupted PD-L1 binding with speckle-type POZ protein, an adaptor …

Identification Of Functional Heterogeneity Of Carcinoma-Associated Fibroblasts With Distinct Il6-Mediated Therapy Resistance In Pancreatic Cancer, Kathleen M Mcandrews, Yang Chen, J Kebbeh Darpolor, Xiaofeng Zheng, Sujuan Yang, Julienne L Carstens, Bingrui Li, Huamin Wang, Toru Miyake, Pedro Correa De Sampaio, Michelle L Kirtley, Mariangela Natale, Chia-Chin Wu, Hikaru Sugimoto, Valerie S Lebleu, Raghu Kalluri

Faculty, Staff and Student Publications

The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) involves a significant accumulation of fibroblasts as part of the host response to cancer. Employing single-cell RNA-sequencing, multiplex immunostaining, and several genetic mouse models, we identify carcinoma-associated fibroblasts (CAFs) with opposing functions in PDAC progression. Depletion of fibroblast activation protein (FAP)+ CAFs results in increased survival, in contrast to depletion of alpha smooth muscle actin (αSMA)+ CAFs that leads to decreased survival. Tumor-promoting FAP+ CAFs (TP-CAFs) and tumor-restraining αSMA+ CAFs (TR-CAFs) differentially regulate cancer-associated pathways and accumulation of Tregs. Improved efficacy of gemcitabine is observed when IL-6 is deleted from αSMA+ CAFs …

Experimental Models Of Undifferentiated Pleomorphic Sarcoma And Malignant Peripheral Nerve Sheath Tumor, Angela D Bhalla, Sharon M Landers, Anand K Singh, Jace P Landry, Michelle G Yeagley, Gabryella S B Myerson, Cristian B Delgado-Baez, Stephanie Dunnand, Theresa Nguyen, Xiaoyan Ma, Svetlana Bolshakov, Brian A Menegaz, Salah-Eddine Lamhamedi-Cherradi, Xizeng Mao, Xingzhi Song, Alexander J Lazar, Ian E Mccutcheon, John M Slopis, Joseph A Ludwig, Dina C Lev, Kunal Rai, Keila E Torres

Faculty, Staff and Student Publications

Undifferentiated pleomorphic sarcoma (UPS) and malignant peripheral nerve sheath tumor (MPNST) are aggressive soft tissue sarcomas that do not respond well to current treatment modalities. The limited availability of UPS and MPNST cell lines makes it challenging to identify potential therapeutic targets in a laboratory setting. Understanding the urgent need for improved treatments for these tumors and the limited cellular models available, we generated additional cell lines to study these rare cancers. Patient-derived tumors were used to establish 4 new UPS models, including one radiation-associated UPS-UPS271.1, UPS511, UPS0103, and RIS620, one unclassified spindle cell sarcoma-USC060.1, and 3 new models of …

Gli1 Activates Pro-Fibrotic Pathways In Myelofibrosis Fibrocytes, Taghi Manshouri, Ivo Veletic, Ping Li, C Cameron Yin, Sean M Post, Srdan Verstovsek, Zeev Estrov

Faculty, Staff and Student Publications

Bone marrow (BM) fibrosis was thought to be induced exclusively by mesenchymal stromal cells (MSCs). However, we and others found that neoplastic fibrocytes induce BM fibrosis in myelofibrosis (MF). Because glioma-associated oncogene-1 (GLI1), an effector of the Hedgehog pathway, plays a role in the induction of BM fibrosis, we wondered whether GLI1 affects fibrocyte-induced BM fibrosis in MF. Multiplexed fluorescence immunohistochemistry analysis of MF patients' BM detected high levels of GLI1 in MF fibrocytes compared to MSCs or normal fibrocytes. Immunostaining, RNA in situ hybridization, gene expression analysis, and western immunoblotting detected high levels of GLI1 and GLI1-induced matrix metalloproteases …

Rapid Acceleration Of Kras-Mutant Pancreatic Carcinogenesis Via Remodeling Of Tumor Immune Microenvironment By Pparδ, Yi Liu, Yasunori Deguchi, Daoyan Wei, Fuyao Liu, Micheline J Moussalli, Eriko Deguchi, Donghui Li, Huamin Wang, Lovie Ann Valentin, Jennifer K Colby, Jing Wang, Xiaofeng Zheng, Haoqiang Ying, Mihai Gagea, Baoan Ji, Jiaqi Shi, James C Yao, Xiangsheng Zuo, Imad Shureiqi

Faculty, Staff and Student Publications

Pancreatic intraepithelial neoplasia (PanIN) is a precursor of pancreatic ductal adenocarcinoma (PDAC), which commonly occurs in the general populations with aging. Although most PanIN lesions (PanINs) harbor oncogenic KRAS mutations that initiate pancreatic tumorigenesis; PanINs rarely progress to PDAC. Critical factors that promote this progression, especially targetable ones, remain poorly defined. We show that peroxisome proliferator-activated receptor-delta (PPARδ), a lipid nuclear receptor, is upregulated in PanINs in humans and mice. Furthermore, PPARδ ligand activation by a high-fat diet or GW501516 (a highly selective, synthetic PPARδ ligand) in mutant KRASG12D (KRASmu) pancreatic epithelial cells strongly accelerates PanIN progression to PDAC. This …

Biliverdin Reductase Bridges Focal Adhesion Kinase To Src To Modulate Synaptic Signaling, Chirag Vasavda, Evan R Semenza, Jason Liew, Ruchita Kothari, Ryan S Dhindsa, Shruthi Shanmukha, Anthony Lin, Robert Tokhunts, Cristina Ricco, Adele M Snowman, Lauren Albacarys, Francesco Pastore, Cristian Ripoli, Claudio Grassi, Eugenio Barone, Michael D Kornberg, Xinzhong Dong, Bindu D Paul, Solomon H Snyder

Duncan NRI Faculty and Staff Publications

Synapses connect discrete neurons into vast networks that send, receive, and encode diverse forms of information. Synaptic function and plasticity—the neuronal process of adapting to diverse and variable inputs—depend on the dynamic nature of synaptic molecular components, which is mediated in part by cell adhesion signaling pathways. Here, we found that the enzyme biliverdin reductase (BVR) physically links together key focal adhesion signaling molecules at the synapse. BVR-null (BVR−/−) mice exhibited substantial deficits in learning and memory on neurocognitive tests, and hippocampal slices in which BVR was postsynaptically depleted showed deficits in electrophysiological responses to stimuli. …

Bhlhe40 Regulates The T-Cell Effector Function Required For Tumor Microenvironment Remodeling And Immune Checkpoint Therapy Efficacy, Avery J Salmon, Alexander S Shavkunov, Qi Miao, Nicholas N Jarjour, Sunita Keshari, Ekaterina Esaulova, Charmelle D Williams, Jeffrey P Ward, Anna M Highsmith, Josué E Pineda, Reshma Taneja, Ken Chen, Brian T Edelson, Matthew M Gubin

Faculty, Staff and Student Publications

Immune checkpoint therapy (ICT) using antibody blockade of programmed cell death protein 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) can provoke T cell-dependent antitumor activity that generates durable clinical responses in some patients. The epigenetic and transcriptional features that T cells require for efficacious ICT remain to be fully elucidated. Herein, we report that anti-PD-1 and anti-CTLA-4 ICT induce upregulation of the transcription factor BHLHE40 in tumor antigen-specific CD8+ and CD4+ T cells and that T cells require BHLHE40 for effective ICT in mice bearing immune-edited tumors. Single-cell RNA sequencing of intratumoral immune cells in BHLHE40-deficient mice revealed differential …

Cross-Species Genetic Screens Identify Transglutaminase 5 As A Regulator Of Polyglutamine-Expanded Ataxin-1, Won-Seok Lee, Ismael Al-Ramahi, Hyun-Hwan Jeong, Youjin Jang, Tao Lin, Carolyn J Adamski, Laura A Lavery, Smruti Rath, Ronald Richman, Vitaliy V Bondar, Elizabeth Alcala, Jean-Pierre Revelli, Harry T Orr, Zhandong Liu, Juan Botas, Huda Y Zoghbi

Duncan NRI Faculty and Staff Publications

Many neurodegenerative disorders are caused by abnormal accumulation of misfolded proteins. In spinocerebellar ataxia type 1 (SCA1), accumulation of polyglutamine-expanded (polyQ-expanded) ataxin-1 (ATXN1) causes neuronal toxicity. Lowering total ATXN1, especially the polyQ-expanded form, alleviates disease phenotypes in mice, but the molecular mechanism by which the mutant ATXN1 is specifically modulated is not understood. Here, we identified 22 mutant ATXN1 regulators by performing a cross-species screen of 7787 and 2144 genes in human cells and Drosophila eyes, respectively. Among them, transglutaminase 5 (TG5) preferentially regulated mutant ATXN1 over the WT protein. TG enzymes catalyzed cross-linking of ATXN1 in a polyQ-length–dependent manner, …

The Microrna-183/96/182 Cluster Inhibits Lung Cancer Progression And Metastasis By Inducing An Interleukin-2-Mediated Antitumor Cd8+ Cytotoxic T-Cell Response, Samrat T Kundu, B Leticia Rodriguez, Laura A Gibson, Amanda N Warner, Mabel G Perez, Rakhee Bajaj, Jared J Fradette, Caleb A Class, Luisa M Solis, Frank R Rojas Alvarez, Ignacio I Wistuba, Lixia Diao, Fengju Chen, Mohit Sachdeva, Jing Wang, David G Kirsch, Chad J Creighton, Don L Gibbons

Faculty, Staff and Student Publications

Here, Kundu et al. investigated the role of the microRNA-183/96/182 cluster (m96cl) in lung cancer and used a novel conditional m96cl mouse to establish that loss of m96cl accelerated the growth of K-Ras mutant autochthonous lung adenocarcinomas. Overall, the authors identified a novel mechanistic role of the m96cl in the suppression of lung cancer growth and metastasis by inducing an IL2-mediated systemic CD8+ CTL immune response.

Reduction Of Mutant Atxn1 Rescues Premature Death In A Conditional Sca1 Mouse Model, James P Orengo, Larissa Nitschke, Meike E Van Der Heijden, Nicholas A Ciaburri, Harry T Orr, Huda Y Zoghbi

Duncan NRI Faculty and Staff Publications

Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disorder. As disease progresses, motor neurons are affected, and their dysfunction contributes toward the inability to maintain proper respiratory function, a major driving force for premature death in SCA1. To investigate the isolated role of motor neurons in SCA1, we created a conditional SCA1 (cSCA1) mouse model. This model suppresses expression of the pathogenic SCA1 allele with a floxed stop cassette. cSCA1 mice crossed to a ubiquitous Cre line recapitulate all the major features of the original SCA1 mouse model; however, they took twice as long to develop. We found that …

Dynamics Of Huntingtin Protein Interactions In The Striatum Identifies Candidate Modifiers Of Huntington Disease, Todd M Greco, Christopher Secker, Eduardo Silva Ramos, Joel D Federspiel, Jeh-Ping Liu, Alma M Perez, Ismael Al-Ramahi, Jeffrey P Cantle, Jeffrey B Carroll, Juan Botas, Scott O Zeitlin, Erich E Wanker, Ileana M Cristea

Duncan NRI Faculty and Staff Publications

Huntington’s disease (HD) is a monogenic neurodegenerative disorder with one causative gene, huntingtin (HTT). Yet, HD pathobiology is multifactorial, suggesting that cellular factors influence disease progression. Here, we define HTT protein-protein interactions (PPIs) perturbed by the mutant protein with expanded polyglutamine in the mouse striatum, a brain region with selective HD vulnerability. Using metabolically labeled tissues and immunoaffinity purification-mass spectrometry, we establish that polyglutamine-dependent modulation of HTT PPI abundances and relative stability starts at an early stage of pathogenesis in a Q140 HD mouse model. We identify direct and indirect PPIs that are also genetic disease modifiers using in-cell two-hybrid …

Ndrg1 In Aggressive Breast Cancer Progression And Brain Metastasis, Emilly S Villodre, Xiaoding Hu, Bedrich L Eckhardt, Richard Larson, Lei Huo, Ester C Yoon, Yun Gong, Juhee Song, Shuying Liu, Naoto T Ueno, Savitri Krishnamurthy, Stefan Pusch, Debu Tripathy, Wendy A Woodward, Bisrat G Debeb

Faculty, Staff and Student Publications

BACKGROUND: N-Myc downstream regulated gene 1 (NDRG1) suppresses metastasis in many human malignancies, including breast cancer, yet has been associated with worse survival in patients with inflammatory breast cancer. The role of NDRG1 in the pathobiology of aggressive breast cancers remains elusive.

METHODS: To study the role of NDRG1 in tumor growth and brain metastasis in vivo, we transplanted cells into cleared mammary fat pads or injected them in tail veins of SCID/Beige mice (n = 7-10 per group). NDRG1 protein expression in patient breast tumors (n = 216) was assessed by immunohistochemical staining. Kaplan-Meier method with 2-sided log-rank test …

Exploiting 4–1bb Immune Checkpoint To Enhance The Efficacy Of Oncolytic Virotherapy For Diffuse Intrinsic Pontine Gliomas, Virginia Laspidea, Montserrat Puigdelloses, Sara Labiano, Lucía Marrodán, Marc Garcia-Moure, Marta Zalacain, Marisol Gonzalez-Huarriz, Naiara Martínez-Vélez, Iker Ausejo-Mauleon, Daniel De La Nava, Guillermo Herrador-Cañete, Javier Marco-Sanz, Elisabeth Guruceaga, Carlos E De Andrea, María Villalba, Oren Becher, Massimo Squatrito, Verónica Matía, Jaime Gállego Pérez-Larraya, Ana Patiño-García, Sumit Gupta, Candelaria Gomez-Manzano, Juan Fueyo, Marta M Alonso

Faculty, Staff and Student Publications

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors, and patient survival has not changed despite many therapeutic efforts, emphasizing the urgent need for effective treatments. Here, we evaluated the anti-DIPG effect of the oncolytic adenovirus Delta-24-ACT, which was engineered to express the costimulatory ligand 4-1BBL to potentiate the antitumor immune response of the virus. Delta-24-ACT induced the expression of functional 4-1BBL on the membranes of infected DIPG cells, which enhanced the costimulation of CD8+ T lymphocytes. In vivo, Delta-24-ACT treatment of murine DIPG orthotopic tumors significantly improved the survival of treated mice, leading to long-term survivors that developed …

Disrupting The Myc-Tfeb Circuit Impairs Amino Acid Homeostasis And Provokes Metabolic Anergy, Mario R Fernandez, Franz X Schaub, Chunying Yang, Weimin Li, Seongseok Yun, Stephanie K Schaub, Frank C Dorsey, Min Liu, Meredith A Steeves, Andrea Ballabio, Alexandar Tzankov, Zhihua Chen, John M Koomen, Anders E Berglund, John L Cleveland

Duncan NRI Faculty and Staff Publications

MYC family oncoproteins are regulators of metabolic reprogramming that sustains cancer cell anabolism. Normal cells adapt to nutrient-limiting conditions by activating autophagy, which is required for amino acid (AA) homeostasis. Here we report that the autophagy pathway is suppressed by Myc in normal B cells, in premalignant and neoplastic B cells of Eμ-Myc transgenic mice, and in human MYC-driven Burkitt lymphoma. Myc suppresses autophagy by antagonizing the expression and function of transcription factor EB (TFEB), a master regulator of autophagy. Mechanisms that sustained AA pools in MYC-expressing B cells include coordinated induction of the proteasome and increases in AA …

Pgc1Α/Β Expression Predicts Therapeutic Response To Oxidative Phosphorylation Inhibition In Ovarian Cancer, Carmen Ghilardi, Catarina Moreira-Barbosa, Laura Brunelli, Paola Ostano, Nicolò Panini, Monica Lupi, Alessia Anastasia, Fabio Fiordaliso, Monica Salio, Laura Formenti, Massimo Russo, Edoardo Arrigoni, Ferdinando Chiaradonna, Giovanna Chiorino, Giulio Draetta, Joseph R Marszalek, Christopher P Vellano, Roberta Pastorelli, Mariarosa Bani, Alessandra Decio, Raffaella Giavazzi

Faculty, Staff and Student Publications

Ovarian cancer is the deadliest gynecologic cancer, and novel therapeutic options are crucial to improve overall survival. Here we provide evidence that impairment of oxidative phosphorylation (OXPHOS) can help control ovarian cancer progression, and this benefit correlates with expression of the two mitochondrial master regulators PGC1α and PGC1β. In orthotopic patient-derived ovarian cancer xenografts (OC-PDX), concomitant high expression of PGC1α and PGC1β (PGC1α/β) fostered a unique transcriptional signature, leading to increased mitochondrial abundance, enhanced tricarboxylic acid cycling, and elevated cellular respiration that ultimately conferred vulnerability to OXPHOS inhibition. Treatment with the respiratory chain complex I inhibitor IACS-010759 caused mitochondrial swelling …

Camk2/Camkii Activates Mlkl In Short-Term Starvation To Facilitate Autophagic Flux, Qionghui Zhan, Jaepyo Jeon, Ying Li, Yu Huang, Jian Xiong, Qiaochu Wang, Tian-Le Xu, Yong Li, Fu-Hai Ji, Guangwei Du, Michael X Zhu

Faculty, Staff and Student Publications

MLKL (mixed lineage kinase domain like pseudokinase) is a well-known core component of necrosome that executes necroptotic cell death upon phosphorylation by RIPK3 (receptor interacting serine/threonine kinase 3). Recent studies also implicate a role of MLKL in endosomal trafficking, which is not always dependent on RIPK3. Using mouse Neuro-2a and L929 as well as human HEK293 and HT29 cells, we show here that MLKL is phosphorylated in response to serum and amino acid deprivation from the culture medium, in a manner that depends on CAMK2/CaMKII (calcium/calmodulin dependent protein kinase II) but not RIPK3. The starvation-induced increase in MLKL phosphorylation was …

Alterations Of The Mdm2 C-Terminus Differentially Impact Its Function In Vivo, Vinod Pant, Neeraj K Aryal, Shunbin Xiong, Gilda P Chau, Natalie W Fowlkes, Guillermina Lozano

Faculty, Staff and Student Publications

Murine double minute 2 (Mdm2) is the principal E3-ubiquitin ligase for p53 and contains a C2H2C4 type RING domain wherein the last cysteine residue is followed by an evolutionarily conserved 13 amino acid C-terminal tail. Previous studies have indicated that integrity of the C-terminal tail is critical for Mdm2 function. Recently, a mutation extending the MDM2 length by five amino acids was identified and associated with enhanced p53 response in fibroblasts and premature aging in a human patient. To investigate the importance of the conserved Mdm2 C-terminal length on p53 regulatory function in vivo, we engineered three novel mouse alleles …

Loss Of Rnf43 Accelerates Kras-Mediated Neoplasia And Remodels The Tumor Immune Microenvironment In Pancreatic Adenocarcinoma, Abdel Nasser Hosein, Gita Dangol, Takashi Okumura, Jason Roszik, Kimal Rajapakshe, Megan Siemann, Mohamed Zaid, Bidyut Ghosh, Maria Monberg, Paola A Guerrero, Aatur Singhi, Cara L Haymaker, Hans Clevers, Lotfi Abou-Elkacem, Sonja M Woermann, Anirban Maitra

Faculty, Staff and Student Publications

Background & aims: RNF43 is an E3 ubiquitin ligase that is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC) and precursor cystic neoplasms of the pancreas. The impact of RNF43 mutations on PDAC is poorly understood and autochthonous models have not been characterized sufficiently. In this study, we describe a genetically engineered mouse model (GEMM) of PDAC with conditional expression of oncogenic Kras and deletion of the catalytic domain of Rnf43 in exocrine cells.

Methods: We generated Ptf1a-Cre;LSL-KrasG12D;Rnf43flox/flox (KRC) and Ptf1a-Cre; LSL-KrasG12D (KC) mice and animal survival was assessed. KRC mice were sacrificed at 2 months, 4 months, and at moribund …

Daam2 Regulates Myelin Structure And The Oligodendrocyte Actin Cytoskeleton Through Rac1 And Gelsolin, Carlo D Cristobal, Chih-Yen Wang, Zhongyuan Zuo, Joshua A Smith, Aaron Lindeke-Myers, Hugo J Bellen, Hyun Kyoung Lee

Duncan NRI Faculty and Staff Publications

Myelin is essential to neuronal health and CNS function, and oligodendrocytes (OLs) undergo a complex process of cytoskeletal remodeling to form compact myelin sheaths. We previously discovered that a formin protein, Dishevelled associated activator of morphogenesis 2 (Daam2), suppresses OL differentiation through Wnt signaling; however, its role in cytoskeletal control remains unknown. To investigate this, we used OL-specific Daam2 conditional knockout (Daam2 cKO) mice of either sex and found myelin decompaction during an active period of myelination in postnatal development and motor coordination deficits in adulthood. Using primary OL cultures, we found Daam2-depleted OLs showed morphologic dysregulation during differentiation, suggesting …

Clinical Exome Sequencing Data Reveal High Diagnostic Yields For Congenital Diaphragmatic Hernia Plus (Cdh+) And New Phenotypic Expansions Involving Cdh, Tiana M Scott, Ian M Campbell, Andres Hernandez-Garcia, Seema R Lalani, Pengfei Liu, Chad A Shaw, Jill A Rosenfeld, Daryl A Scott

Duncan NRI Faculty and Staff Publications

Background: Congenital diaphragmatic hernia (CDH) is a life-threatening birth defect that often co-occurs with non-hernia-related anomalies (CDH+). While copy number variant (CNV) analysis is often employed as a diagnostic test for CDH+, clinical exome sequencing (ES) has not been universally adopted.

Methods: We analysed a clinical database of ~12 000 test results to determine the diagnostic yields of ES in CDH+ and to identify new phenotypic expansions.

Results: Among the 76 cases with an indication of CDH+, a molecular diagnosis was made in 28 cases for a diagnostic yield of 37% (28/76). A provisional diagnosis was made in seven other …

Inhibition Of Calcium-Triggered Secretion By Hydrocarbon-Stapled Peptides, Ying Lai, Giorgio Fois, Jose R Flores, Michael J Tuvim, Qiangjun Zhou, Kailu Yang, Jeremy Leitz, John Peters, Yunxiang Zhang, Richard A Pfuetzner, Luis Esquivies, Philip Jones, Manfred Frick, Burton F Dickey, Axel T Brunger

Faculty, Staff and Student Publications

Membrane fusion triggered by Ca2+ is orchestrated by a conserved set of proteins to mediate synaptic neurotransmitter release, mucin secretion and other regulated exocytic processes1-4. For neurotransmitter release, the Ca2+ sensitivity is introduced by interactions between the Ca2+ sensor synaptotagmin and the SNARE complex5, and sequence conservation and functional studies suggest that this mechanism is also conserved for mucin secretion6. Disruption of Ca2+-triggered membrane fusion by a pharmacological agent would have therapeutic value for mucus hypersecretion as it is the major cause of airway obstruction in the pathophysiology of respiratory viral infection, asthma, chronic obstructive pulmonary disease and cystic fibrosis7-11. …

Characterization Of Patient-Derived Bone Marrow Human Mesenchymal Stem Cells As Oncolytic Virus Carriers For The Treatment Of Glioblastoma, Yuzaburo Shimizu, Joy Gumin, Feng Gao, Anwar Hossain, Elizabeth J Shpall, Akihide Kondo, Brittany C Parker Kerrigan, Jing Yang, Daniel Ledbetter, Juan Fueyo, Candelaria Gomez-Manzano, Frederick F Lang

Faculty, Staff and Student Publications

Objective: Delta-24-RGD is an oncolytic adenovirus that is capable of replicating in and killing human glioma cells. Although intratumoral delivery of Delta-24-RGD can be effective, systemic delivery would improve its clinical application. Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) obtained from healthy donors have been investigated as virus carriers. However, it is unclear whether BM-hMSCs can be derived from glioma patients previously treated with marrow-toxic chemotherapy or whether such BM-hMSCs can deliver oncolytic viruses effectively. Herein, the authors undertook a prospective clinical trial to determine the feasibility of obtaining BM-hMSCs from patients with recurrent malignant glioma who were previously exposed …

Local Treatment Of A Pediatric Osteosarcoma Model With A 4-1bbl Armed Oncolytic Adenovirus Results In An Antitumor Effect And Leads To Immune Memory, Naiara Martinez-Velez, Virginia Laspidea, Marta Zalacain, Sara Labiano, Marc García-Moure, Montse Puigdelloses, Lucía Marrodan, Marisol Gonzalez-Huarriz, Guillermo Herrador, Daniel De La Nava, Iker Ausejo-Mauleon, Juan Fueyo, Candelaria Gomez-Manzano, Ana Patiño-García, Marta M Alonso

Faculty, Staff and Student Publications

Osteosarcoma is an aggressive bone tumor occurring primarily in pediatric patients. Despite years of intensive research, the outcomes of patients with metastatic disease or those who do not respond to therapy have remained poor and have not changed in the last 30 years. Oncolytic virotherapy is becoming a reality to treat local and metastatic tumors while maintaining a favorable safety profile. Delta-24-ACT is a replicative oncolytic adenovirus engineered to selectively target cancer cells and to potentiate immune responses through expression of the immune costimulatory ligand 4-1BB. This work aimed to assess the antisarcoma effect of Delta-24-ACT. MTS and replication assays …