Medical Genetics Commons^™

Tubulin Regulates Stability And Localization Of Stmn2 By Binding Preferentially To Its Soluble Form, Xiang Deng, Gary A Bradshaw, Marian Kalocsay, Timothy Mitchison

Faculty, Staff and Student Publications

The small, tubulin-binding protein STMN2 is highly expressed in neurons and is implicated in amyotrophic lateral sclerosis. STMN2 degrades rapidly and accumulates at axotomy sites, suggesting fast turnover is crucial for its neuroprotective function. We show that STMN2 was primarily degraded by the ubiquitin-proteasome system. Its membrane-targeting N-terminal domain promoted fast turnover, whereas its tubulin-binding domain promoted stabilization. Proximity labeling and imaging showed that tubulin binding reduced STMN2 targeting to trans-Golgi network membranes. Pull-down assays showed that tubulin binds preferentially to soluble over membrane-bound STMN2. Our observations suggest that STMN2 interconverts between a soluble, tubulin-bound form and a membrane-bound, tubulin-free …

Secretory Iga Dysfunction Underlies Poor Prognosis In Fusobacterium-Infected Colorectal Cancer, Ilseok Choi, Kyung-A Kim, Sang Cheol Kim, Donghwan Park, Ki Taek Nam, Jun Hyung Cha, Seungbyn Baek, Junha Cha, Hye-Yeong Jo, Minsun Jung, Melody Y Zeng, Irina Matei, Susan Bullman, Joong Bae Ahn, Yoon Dae Han, Han Sang Kim, Insuk Lee

Faculty, Staff and Student Publications

Fusobacterium nucleatum (Fn) is commonly enriched in colorectal cancer (CRC) and associated with poor outcomes, though its mechanisms remain unclear. Our study investigated how Fn affects the tumor microenvironment through single-cell transcriptomic analyses of 42 CRC patient tissues, comparing Fn-positive and Fn-negative tumors. We discovered that Fn impairs IgA plasma cell development and secretory IgA (sIgA) production by disrupting communication with tumor-associated macrophages. Additional experiments in germ-free mice, together with our re-analysis of a publicly available single-cell RNA-seq data set from a CRC mouse model with an intact gut microbiome–both models having been orally gavaged with Fn–jointly validated the causal …

Loss Of The Lysosomal Protein Cln3 Triggers C-Abl-Dependent Yap1 Pro-Apoptotic Signaling, Neuza Domingues, Alessia Calcagni', Sofia Freire, Joana Pires, Ricardo Casqueiro, Ivan L Salazar, Niculin Joachim Herz, Tuong Huynh, Katarzyna Wieciorek, Tiago Fleming Outeiro, Henrique Girão, Ira Milosevic, Andrea Ballabio, Nuno Raimundo

Duncan NRI Faculty and Staff Publications

Batten disease is characterized by early-onset blindness, juvenile dementia and death within the second decade of life. The most common genetic cause are mutations in CLN3, encoding a lysosomal protein. Currently, no therapies targeting disease progression are available, largely because its molecular mechanisms remain poorly understood. To understand how CLN3 loss affects cellular signaling, we generated human CLN3 knock-out cells (CLN3-KO) and performed RNA-seq analysis. Our multi-dimensional analysis reveals the transcriptional regulator YAP1 as a key factor in remodeling the transcriptome in CLN3-KO cells. YAP1-mediated pro-apoptotic signaling is also increased as a consequence of CLN3 functional loss in retinal pigment …

From Bladder To Brain: How You Know When It’S Time To Go, Anne M J Verstegen, Kara L Marshall

Duncan NRI Faculty and Staff Publications

The decision to urinate relies on assessing bladder fullness and context to determine an appropriate time and place to go. Any disruption in this interoceptive process results in frequent and sometimes debilitating consequences in daily life. Recent work has uncovered key pathways and brain regions that contribute to the sense of bladder stretch and the control of urinary reflexes, but many open questions remain. Here, we review the known mechanisms that convey sensory information from the bladder to the brain and back down again, and we highlight the knowledge gaps and opportunities for better understanding this system, which will be …

Developmental Stage-Dependent Transcriptomic Responses To Neonatal Intraventricular Hemorrhage, Elizabeth Wallace-Anthony, Miriam Zamorano, Hemendra J Vekaria, Braden B Oldham, Kiara P Umpornpun, Scott D Olson, Stefano Berto, Brandon A Miller

Faculty, Staff and Student Publications

Neonatal intraventricular hemorrhage (IVH) is a major complication of preterm birth, yet how developmental stage influences the brain's response to injury remains unclear. We performed single-nucleus RNA sequencing on rat brains 24 h after IVH at postnatal day 2 (PND2) or day 5 (PND5) to define transcriptional responses across cell types. We identified 42 distinct cell populations and found that PND5 brains exhibited a markedly stronger immune and inflammatory response to IVH, with a threefold increase in differentially expressed genes compared to PND2. Microglia were the most perturbed cell type at both stages, showing increased oxidative stress and polarization toward …

Mecp2 Interacts With The Super Elongation Complex To Regulate Transcription, Jun Young Sonn, Wonho Kim, Marta Iwanaszko, Yuki Aoi, Yan Li, Guantong Qi, Luke Parkitny, Janice L Brissette, Lorin Weiner, Juan Botas, Ismael Al-Ramahi, Ali Shilatifard, Huda Y Zoghbi

Faculty, Staff and Students Publications

Loss-of-function mutations in methyl-CpG binding protein 2 (MECP2) cause Rett syndrome. While we know that MeCP2 binds to methylated cytosines on DNA, the full breadth of the molecular mechanisms by which MeCP2 regulates gene expression remains incompletely understood. Here, using a genetic modifier screen, we identify the super elongation complex, a P-TEFb–containing elongation factor that releases promoter-proximally paused RNA polymerase II, as a genetic interactor of MECP2. MeCP2 physically interacts with SEC subunits and directly binds AFF4, the scaffold of the SEC, via the transcriptional repression domain. Furthermore, MeCP2 facilitates the binding of AFF4 on a subset …

Investigating The Neuronal Role Of The Proteasomal Atpase Subunit Gene Psmc5 In Neurodevelopmental Proteasomopathies, Sébastien Küry, Janelle E Stanton, Geeske M Van Woerden, Amélie Bosc-Rosati, Tzung-Chien Hsieh, Lise Bray, Marielle Oloudé, Cory Rosenfelt, Marie Pier Scott-Boyer, Victoria Most, Tianyun Wang, Jonas J Papendorf, Charlotte De Konink, Wallid Deb, Virginie Vignard, Maja Studencka-Turski, Thomas Besnard, Anna M Hajdukowicz, Franziska G Thiel, Sophie Wolfgramm, Laëtitia Florenceau, Silvestre Cuinat, Sylvain Marsac, Yann Verrès, Audrey Dangoumau, Léa Poirier, Ingrid M Wentzensen, Annabelle Tuttle, Cara Forster, Johanna Striesow, Richard Golnik, Damara Ortiz, Laura Jenkins, Jill A Rosenfeld, Alban Ziegler, Clara Houdayer, Dominique Bonneau, Erin Torti, Amber Begtrup, Kristin G Monaghan, Sureni V Mullegama, Catharina M L Nienke Volker-Touw, Koen L I Van Gassen, Renske Oegema, Mirjam S De Pagter, Katharina Steindl, Anita Rauch, Ivan Ivanovski, Kimberly Mcdonald, Emily Boothe, Andrew Dauber, Janice Baker, Noelle Andrea V Fabie, Raphael A Bernier, Tychele N Turner, Siddharth Srivastava, Kira A Dies, Lindsay C Swanson, Carrie Costin, Alali Abdulrazak, Rebekah K Jobling, John Pappas, Rachel Rabin, Dmitriy Niyazov, Anne Chun-Hui Tsai, Karen Kovak, David B Beck, May Christine V Malicdan, David R Adams, Lynne Wolfe, Rebecca D Ganetzky, Colleen C Muraresku, Davit Babikyan, Zdeněk Sedláček, Miroslava Hančárová, Andrew T Timberlake, Hind Al Saif, Berkley Nestler, Kayla King, M J Hajianpour, Gregory Costain, D'Arcy Prendergast, Chumei Li, David Geneviève, Antonio Vitobello, Arthur Sorlin, Christophe Philippe, Tamar Harel, Ori Toker, Ataf Sabir, Derek Lim, Mark J Hamilton, Lisa J Bryson, Elaine Cleary, Sacha Weber, Trevor L Hoffman, Anna M Cueto-González, Eduardo F Tizzano, David Gómez-Andrés, Marta Codina-Solà, Athina Ververi, Efterpi Pavlidou, Alexandros Lambropoulos, Kyriakos Garganis, Marlène Rio, Jonathan Levy, Sarah J Langas, Anne M Mcrae, Mathieu K Lessard, Maria Daniela D'Agostino, Isabelle De Bie, Meret Wegler, Rami Abou Jamra, Susanne B Kamphausen, Viktoria Bothe, Lorraine Potocki, Eric Olinger, Yves Sznajer, Elsa Wiame, Michelle L Thompson, Molly C Schroeder, Catherine Gooch, Raphael A Smith, Arti Pandya, Larissa M Busch, Uwe Völker, Elke Hammer, Kristian Wende, Benjamin Cogné, Bertrand Isidor, Jens Meiler, Clémentine Ripoll, Stéphanie Bigou, Frédéric Laumonnier, Peter W Hildebrand, Evan E Eichler, Kirsty Mcwalter, Peter M Krawitz, Florence Roux-Dalvai, Ype Elgersma, Julien Marcoux, Marie-Pierre Bousquet, Arnaud Droit, Jeremie Poschmann, Andreas M Grabrucker, Francois V Bolduc, Stéphane Bézieau, Frédéric Ebstein, Elke Krüger

Faculty, Staff and Students Publications

Neurodevelopmental proteasomopathies are a group of disorders caused by variants in proteasome subunit genes, that disrupt protein homeostasis and brain development through poorly characterized mechanisms. Here, we report 26 distinct variants in PSMC5, encoding the AAA⁺ ATPase subunit PSMC5/RPT6, in individuals with syndromic neurodevelopmental conditions. Combining genetic, multi-omics and biochemical approaches across cellular models and Drosophila, we unveil the essential role of proteasomes in sustaining key cellular processes. Loss of PSMC5/RPT6 function impairs proteasome activity, leading to protein aggregation, disruption of mitochondrial homeostasis, and dysregulation of lipid metabolism and immune signaling. It also compromises synaptic balance, neuritogenesis, and neural progenitor …

Targeting The Hepatic Circadian Clock Concomitant With Tyrosine Kinase Inhibition Reverses Late-Stage Hepatocellular Carcinoma, Baharan Fekry, Savera Aggarwal, Rachel Van Drunen, Rafael Bravo, Andy Escalante, Constance Atkins, Sheng Pan, Zheng Chen, Kai Sun, David R Hall, Mamoun Younes, Kristin Eckel-Mahan

Faculty, Staff and Student Publications

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Most patients present at advanced stages, and the effectiveness of tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors is constrained by limited patient response. A subset of HCC shows elevated expression of the promoter 2 ("P2")-driven hepatocyte nuclear factor 4 alpha (HNF4α) isoform, which directly transcriptionally represses the circadian brain and muscle ARNT-like protein 1 (BMAL1) transcription factor. This subtype of HCC is robustly inhibited by the plant-based flavonoid nobiletin (NOB), a circadian-fortifying compound. Using patient-matched human HCC and serum, we show that BMAL1-deficient HCC shows exaggerated carnitine palmitoyl transferase …

Live-Cell Quantitative Monitoring Reveals Distinct, High-Affinity Gβγ Regulations Of Girk2 And Girk1/2 Channels, Reem Handklo-Jamal, Tal Keren Raifman, Boris Shalomov, Patrick Hofer, Uri Kahanovitch, Theres Friesacher, Galit Tabak, Vladimir Tsemakhovich, Haritha P Reddy, Orna Chomsky-Hecht, Debi Ranjan Tripathy, Kerstin Zuhlke, Carmen W Dessauer, Enno Klussmann, Yoni Haitin, Joel A Hirsch, Anna Stary-Weinzinger, Daniel Yakubovich, Nathan Dascal

Faculty, Staff and Student Publications

Gi/o protein-coupled receptors (GPCRs) inhibit cardiac and neuronal excitability via G protein-activated K+ channels (GIRK), assembled by combinations of GIRK1 - GIRK4 subunits. GIRKs are activated by direct binding of the Gβγ dimer of inhibitory Gi/o proteins. However, key aspects of this textbook signaling pathway remain debated. Recent studies suggested no Gi/o-GIRK pre-coupling and low (>250 µM) Gβγ-GIRK interaction affinity, contradicting earlier sub-µM estimates and implying low signaling efficiency. We show that Gγ prenylation, which mediates Gβγ membrane attachment required for GIRK activation, also contributes to the Gβγ-GIRK interaction, explaining the poor affinity obtained with non-prenylated Gβγ. Using quantitative …

Naphthalimide-Based Type-I Nano-Photosensitizers For Enhanced Antitumor Photodynamic Therapy: H2s Synergistically Regulates Pet And Self-Assembly, Huiyu Niu, Songnan Wang, Yang Liu, Nana Ma, Shuaiwei Cheng, Beidou Feng, Hyunsun Jeong, Yonggang Yang, Ge Wang, Tony D James, Juyoung Yoon, Jonathan L Sessler, Hua Zhang

Faculty, Staff and Student Publications

Photodynamic therapy (PDT) relies on a combination of light and photosensitizers (PSs) to achieve local control over cancerous lesions. However, it is subject to limitations, including tumor hypoxia, low tumor targeting, off‐target phototoxicity, and always‐on fluorescence. Here, we propose a design strategy for activated nano‐PSs (N‐PSs) to simultaneously overcome the limitations of PDT, wherein photoinduced electron transfer (PeT) is coupled with an endogenous H2S‐regulated self‐association process to promote Type‐I photochemical reactions. Using theoretical calculations, spectral analysis, and microscopic imaging, we verified the generation of self‐assembly and occurrence of PeT. And it was also shown that H2S could synergistically inhibit the …

Divergent Prefrontal Cortex Circuits Regulate Cued Food Seeking Under Distinct Metabolic Or Emotional States, Xu O Zhang, Guillermo Aquino-Miranda, Claire E Cho, Yongzhe Wang, Duy Hoang Ha, Nikita Elinson-Watson, Allen Dong, Caleb Kemere, Fabricio H Do-Monte

Faculty, Staff and Student Publications

Flexibly adjusting food-seeking behaviour in response to food-associated cues and internal states is crucial for animals' survival. However, the neural mechanisms that modulate cued food-seeking behaviour during varying metabolic (i.e., hungry vs. satiated) and emotional (i.e., safe vs. threatened) states remain elusive. Here, we show that the encoding of metabolic or threat states in projection-defined neurons in the prelimbic cortex (PL) mediates cued food-seeking responses in rats. Using microendoscopic imaging, we demonstrate that neural population dynamics in PL consistently represent food cues, task-relevant behaviours, and internal state changes by recruiting distinct subsets of cue-responsive neurons at each state. Single-unit recording …

Tca Cycle Mode Switch Determines The Fate Of Pirtobrutinib-Tolerant Persister Cells In Mantle Cell Lymphoma, Wei Wang, Qingsong Cai, Yang Liu, Lei Nie, Heng-Huan Lee, Fangfang Yan, Yue Fei, Yixin Yao, Yijing Li, Lin Tan, Philip L Lorenzi, Ying-Nai Wang, Jun Yao, Zhihong Chen, Joseph Mitchell Mcintosh, Cheng-Tai Yu, Preetesh Jain, Vivian C Jiang, Jovanny Vargas, Xiaolin Li, Tianci Zhang, Shaoying Li, David Santos, Selvi Thirumurthi, Erin Heather Seeley, Lukas Mikolaj Simon, Christopher Flowers, Chi Young Ok, Michael Wang

Faculty, Staff and Student Publications

Bruton tyrosine kinase inhibitors (BTKis) and cell therapy have successfully been used to treat mantle cell lymphoma (MCL). However, therapy resistance inevitably emerges. Cancer cells can progressively develop stable resistance by traversing through a transient drug-tolerant persister (DTP) state. The mechanisms enabling DTP cells to reversibly adapt to therapies and evolve to acquire heterogeneity remain poorly understood, and characterizing DTP cells in MCL continues to pose a challenge for clinic translation. Here, using pirtobrutinib, a recently US Food and Drug Administration-approved noncovalent BTKi, we identified pirtobrutinib-tolerant persister cells exhibiting morphological variability by presenting a unique population of enlarged cells (giant …

Insights Into The Relevance Of Targeting Fibroblasts To Control Cancer, Viktoria Boeker, Raghu Kalluri

Faculty, Staff and Student Publications

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) have garnered significant research attention in the last decade. As key stromal cells of the TME, studies have explored them as a potential target for controlling cancer. Using high-throughput technologies like single-cell RNA sequencing coupled with proteomics, the classification of different CAF subgroups reveals a complex system that varies by cancer type. Unraveling novel big data, potentially through AI platforms, will be key to identifying the role of CAFs in tumor progression and therapy escape mechanisms, enabling new therapies that manipulate CAFs to increase patients' survival. We summarize and discuss new developments …

Voices: Future Directions In Targeting The Tumor Microenvironment, Tanja De Gruijl, Catherine Sautès-Fridman, Daniela S Thommen, Michael A Curran

Faculty, Staff and Student Publications

What do you envision as the most promising future directions for therapeutic strategies aimed at modulating the tumor microenvironment?

Multiomic Analysis Reveals A Key Bcat1 Role In Mtor Activation By B Cell Receptor And Tlr9, Rui Guo, Yizhe Sun, Matthew Y Lim, Hardik Shah, Joao A Paulo, Rahaman A Ahmed, Weixing Li, Yuchen Zhang, Haopeng Yang, Liang Wei Wang, Daniel Strebinger, Nicholas A Smith, Meng Li, Merrin Man Long Leong, Michael Lutchenkov, Jin Hua Liang, Zhixuan Li, Yin Wang, Rishi Puri, Ari Melnick, Michael R Green, John M Asara, Adonia E Papathanassiu, Duane R Wesemann, Steven P Gygi, Vamsi K Mootha, Benjamin E Gewurz

Faculty, Staff and Student Publications

B lymphocytes play major adaptive immune roles, producing antibodies and driving T cell responses. However, how immunometabolism networks support B cell activation and differentiation in response to distinct receptor stimuli remains incompletely understood. To gain insights, we systematically investigated acute primary human B cell transcriptional, translational, and metabolomic responses to B cell receptor (BCR), TLR9, CD40-ligand (CD40L), IL-4, or combinations thereof. T cell-independent BCR/TLR9 costimulation, which drives malignant and autoimmune B cell states, highly induced transaminase branched chain amino acid transaminase 1 (BCAT1), which localized to lysosomal membranes to support branched chain amino acid synthesis and mTORC1 activation. BCAT1 inhibition …

Multisite Assembly Of Gateway Induced Clones (Magic): A Flexible Cloning Toolbox For Use In Vertebrate Model Systems, William B Gillespie, Yuwen Zhang, Oscar E Ruiz, Juan Cerda, Joshua Ortiz-Guzman, Michelle Sherman, Williamson D Turner, Gabrielle Largoza, Lili E Mosser, Esther Fujimoto, Chi-Bin Chien, Kristen M Kwan, Benjamin R Arenkiel, W Patrick Devine, Joshua D Wythe

Duncan NRI Faculty and Staff Publications

Here, we present MultiSite Assembly of Gateway Induced Clones (MAGIC), which leverages Gateway-based recombinatorial cloning technology for rapid, modular assembly of plasmids to facilitate transgenesis in cells and vertebrate animal models. The MAGIC collection of plasmids spans a range of in vitro and in vivo uses, from tools for optically and chemically tunable gene expression, to simultaneous expression of microRNAs and fluorescent reporters, to a suite of distinct subcellular compartmental fluorescent reporters, to Cre and Dre recombinase-dependent gene expression. MAGIC system components are compatible with existing MultiSite Gateway Tol2 systems currently used in zebrafish and mammalian lentiviral and adenoviral Destination …

Oligomeric Cystatin C Supports The Immunosuppressive Activity Of Myeloid Cells Through Interaction With Inhibitory Receptors, Chengcheng Zhang, Yubo He, Xiaoye Liu, Jingjing Xie, Meng Fang, Xing Yang, Ryan Huang, Qi Lou, Bufan Li, Ankit Gupta, Cheryl Lewis, Marc I Diamond, Ningyan Zhang, Zhiqiang An, Cheng Cheng Zhang

Faculty, Staff and Student Publications

Amyloid proteins are linked to various diseases; however, their functional roles in immunity and cancer remain unclear. Here, we establish a direct link between oligomeric cystatin C-a cysteine cathepsin inhibitor and a well-characterized amyloidogenic protein-within the tumor microenvironment and the immune inhibitory receptors LILRB2 and LILRB5 on myeloid cells. We demonstrated that human LILRB2 and LILRB5, along with their murine counterpart PIRB, serve as functional receptors for cystatin C oligomers. Engagement of these inhibitory receptors by oligomeric cystatin C enhances the immunosuppressive activity of myeloid cells, leading to T-cell suppression and tumor progression. Deletion of the CST3 gene, which encodes …

Developing A General Ai Model For Integrating Diverse Genomic Modalities And Comprehensive Genomic Knowledge, Zhenhao Zhang, Xinyu Bao, Linghua Jiang, Xin Luo, Zheyu Zhang, Jing Yin, Meiqi Zhao, Yichun Wang, Annelise Comai, Joerg Waldhaus, Anders S Hansen, Wenbo Li, Jie Liu

Faculty, Staff and Student Publications

Advances in next-generation sequencing technologies have vastly expanded the availability of diverse genomic, epigenomic, and transcriptomic data, presenting the opportunity to develop a general AI model that integrates comprehensive genomic knowledge into a unified model. Unlike previous predictive models, which are typically specialized to certain tasks, our general AI model unifies a wide range of genomic modalities, such as nascent RNA and ultra-high-resolution chromatin organization, within a multi-task architecture. Using ATAC-seq and DNA sequences as inputs, we incorporated diverse genomic modalities as output, and the model exhibits strong generalizability across different cell types and tissues in all tasks we trained. …

Low Mutation Rate But High Male-Bias In The Germline Of A Short-Lived Opossum, Yadira Peña-García, Richard J Wang, Muthuswamy Raveendran, R Alan Harris, Paul B Samollow, Jeffrey Rogers, Matthew W Hahn

Faculty, Staff and Students Publications

Age and sex have been found to be important determinants of the mutation rate per generation in mammals, but the mechanisms underlying these factors are still unclear. One approach to distinguishing between alternative mechanisms is to study species that reproduce at very young ages, as competing hypotheses make different predictions about patterns of mutation in these organisms. Here, we study the germline mutation rate in the gray short-tailed opossum, Monodelphis domestica, a laboratory model species that becomes reproductively mature at less than 6 mo of age. Whole-genome sequencing of 22 trios reveals one of the lowest mutation rates per generation …

Slc35g3 Is A Udp-N-Acetylglucosamine Transporter For Sperm Glycoprotein Formation And Underpins Male Fertility In Mice, Daisuke Mashiko, Shingo Tonai, Haruhiko Miyata, Martin M Matzuk, Masahito Ikawa

Faculty, Staff and Students Publications

Despite the recognized importance of glycans in biological phenomena, their complex roles in spermatogenesis and sperm function remain unclear. SLC35G3, a 10-transmembrane protein specifically found in early round spermatids, belongs to the sugar-nucleotide transporter family, indicating its involvement in glycan formation. In this study, we found that Slc35g3 knockout male mice were sterile due to impaired sperm functions in uterotubal junction passage, zona pellucida binding, and oocyte fusion. Mouse SLC35G3 has UDP-GlcNAc transporter activity, and its ablation caused abnormal processing of the sperm plasma membrane and acrosome membrane proteins. Reported human SLC35G3 mutations (F267L and T179HfsTer27) diminished the UDP-GlcNAc transporter …

Reprogramming Tumor Microenvironment Via Systemic Delivery Of Tlr3 Agonist And Manganese Nanoparticle, Young Seok Cho, Xingwu Zhou, Xiaoqi Sun, Ziye Wan, Julia Crowther, Mariko Takahashi, Swetha Kodamasimham, Qi Wu, May Thazin Phoo, Youngseo Na, Kai Han, Zaiye Li, Anna Schwendeman, Steven P Schwendeman, Yu Leo Lei, James J Moon

Faculty, Staff and Student Publications

Toll-like receptor (TLR) agonists, as potent immunostimulatory adjuvants, play a critical role in linking the innate and adaptive immune responses. However, their antitumor effects as cancer immunotherapeutic agents have been limited. Here, we report our finding that manganese ion (Mn2+) potentiates various TLR agonists, leading to robust activation of the TLR pathway and the stimulator of interferon genes (STING) pathway among innate immune cells. In particular, we have observed robust antitumor efficacy after intratumoral administration of a TLR3 agonist and Mn2+. To achieve systemic codelivery of TLR3 agonist and Mn2+, we have developed a low-molecular-weight poly(inosinic:cytidylic acid)-Mn2+ coordination lipid nanoparticle …

Genome-Wide Crispr Screens Identify Critical Targets To Enhance Car-Nk Cell Antitumor Potency, Alexander Biederstädt, Rafet Basar, Jeong-Min Park, Nadima Uprety, Rejeena Shrestha, Francia Reyes Silva, Merve Dede, John Watts, Sunil Acharya, Donghai Xiong, Bin Liu, May Daher, Hind Rafei, Pinaki Banerjee, Ping Li, Sanjida Islam, Huihui Fan, Mayra Shanley, Jingling Jin, Bijender Kumar, Vernikka Woods, Paul Lin, Silvia Tiberti, Ana Karen Nunez Cortes, Xin Ru Jiang, Inci Biederstädt, Patrick Zhang, Ye Li, Seema Rawal, Enli Liu, Luis Muniz-Feliciano, Gary M Deyter, Elizabeth J Shpall, Natalie Wall Fowlkes, Ken Chen, Katayoun Rezvani

Faculty, Staff and Student Publications

Adoptive cell therapy using engineered natural killer (NK) cells is a promising approach for cancer treatment, with targeted gene editing offering the potential to further enhance their therapeutic efficacy. However, the spectrum of actionable genetic targets to overcome tumor and microenvironment-mediated immunosuppression remains largely unexplored. We performed multiple genome-wide CRISPR screens in primary human NK cells and identified critical checkpoints regulating resistance to immunosuppressive pressures. Ablation of MED12, ARIH2, and CCNC significantly improved NK cell antitumor activity against multiple treatment-refractory human cancers in vitro and in vivo. CRISPR editing augmented both innate and CAR-mediated NK cell function, associated with enhanced …

Development Of A Targeted Bioprotac Degrader Selective For Misfolded Sod1, Christen G Chisholm, Rachael Bartlett, Mikayla L Brown, Emma-Jayne Proctor, Natalie E Farrawell, Jody Gorman, Fabien Delerue, Lars M Ittner, Kara L Vine-Perrow, Heath Ecroyd, Neil R Cashman, Darren N Saunders, Luke Mcalary, Jeremy S Lum, Justin J Yerbury

Faculty, Staff and Student Publications

The accumulation of misfolded proteins underlies a broad range of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Due to their dynamic nature, these misfolded proteins have proven challenging to target therapeutically. Here, we specifically target misfolded disease variants of the ALS-associated protein superoxide dismutase 1 (SOD1), using a biological proteolysis targeting chimera (BioPROTAC) composed of a SOD1-specific intrabody and an E3 ubiquitin ligase. Screening of intrabodies and E3 ligases for optimal BioPROTAC construction reveals a candidate capable of degrading multiple disease variants of SOD1, preventing their aggregation in cells. Using CRISPR/Cas9 technology to develop a BioPROTAC transgenic mouse line, we …

Cofilin Inhibition Ameliorates Piezo2 And Ampa Dysfunction In A Mouse Model Of Angelman Syndrome, Luis O Romero, Manisha Bade, Elisa Carrillo, Sonia Paz-López, Syed A M Hasan, William James Antonisamy, Vasanthi Jayaraman, Zahoor A Shah, Valeria Vásquez, Julio F Cordero-Morales

Faculty, Staff and Student Publications

Angelman syndrome (AS) is a neurogenetic disorder characterized by motor coordination and cognitive deficits. In AS, hippocampal neurons show reduced filamentous (F-)actin, a decrease we also reported in dorsal root ganglia (DRG) neurons, along with impaired mechanosensitive ion channel activity. Currently, there are no pharmacological targets to prevent the decrease of F-actin in AS. Here, we utilize a first-in-class selective cofilin inhibitor (SZ-3) to restore PIEZO2 function in DRG neurons and glutamate-evoked currents in hippocampal neurons from AS mice. Using atomic force microscopy, we demonstrate that inhibiting cofilin, an actin-severing protein, with SZ-3 increases cellular stiffness by stabilizing the actin …

Preclinical Fluorescence-Guided Imaging Leveraging Surrounding Sentinel Tumor Microenvironment Identifies High-Risk Premalignant Pancreatic Lesions, Shilpa Sharma, Xiaoxia Wen, Jianbo Wang, Beibei Huang, Denise A Hernandez, Cong-Dat Pham, Zhiwen Liu, Susanne Je-Han Lin, Aiko Yamaguchi, Dimitra K Georgiou, Ryan P Coll, H Charles Manning

Faculty, Staff and Student Publications

Purpose: Because surgery is the only potential cure for pancreatic cancer, high-risk premalignant pancreatic lesions often evade detection by palpation or white-light visualization, increasing the risk of recurrence. We asked whether near-infrared fluorescence imaging of tumor-associated inflammation could identify high-risk premalignant lesions, leveraging the tumor microenvironment as a sentinel of local disease and, thus, enhance surgery outcomes.

Experimental design: Fluorescence-guided surgery was performed on genetically engineered mice [Ptf1a-Cre; LSL-KrasG12D/+; Smad4flox/flox (KSC)] at discrete stages of disease progression, histologically confirmed high-risk, premalignant lesions in postnatal mice to locally advanced pancreatic tumors in adults, using the imaging agent V-1520, a translocator protein …

An Annotated Biobank Of Triple-Negative Breast Cancer Patient-Derived Xenografts Features Treatment-Naïve And Longitudinal Samples During Neoadjuvant Chemotherapy, Amanda L Rinkenbaugh, Yuan Qi, Shirong Cai, Jiansu Shao, Faiza Baameur Hancock, Sabrina L Jeter-Jones, Xiaomei Zhang, Emily Powell, Lei Huo, Rosanna Lau, Chunxiao Fu, Rebekah Gould, Petra Den Hollander, Elizabeth E Ravenberg, Jason B White, Gaiane M Rauch, Banu Arun, Clinton Yam, Alastair M Thompson, Gloria V Echeverria, Stacy L Moulder, W Fraser Symmans, Jeffrey T Chang, Helen Piwnica-Worms

Faculty, Staff and Student Publications

Triple-negative breast cancer (TNBC) that fails to respond to neoadjuvant chemotherapy (NACT) can be lethal. Developing effective strategies to eradicate chemoresistant disease requires experimental models that recapitulate the heterogeneity characteristic of TNBC. To that end, we established a biobank of 92 orthotopic patient-derived xenograft (PDX) models of TNBC from the tumors of 75 patients enrolled in the ARTEMIS clinical trial (NCT02276443), including 12 longitudinal sets generated from serial patient biopsies collected throughout NACT treatment and from metastatic disease. Models were established from both chemosensitive and chemoresistant tumors, and nearly 30% of the PDX models were capable of metastasizing …

Apol2 Stabilizes Ku80 To Confer Nhej-Mediated Radioresistance In Gastric Cancer, Dan Zu, Qimei Bao, Hanyi He, Yuke Zhong, Mingcong Deng, Yangchan Hu, Chunkai Zhang, Chen Liang, Yixing Huang, Haidong Liu, Xiao Li, Yanhua He, Guoyan Luo, Weixin Wu, Fenghui Guan, Shengfeng Xu, Min Liu, Albino Bacolla, Ji Jing, Yian Du, John A Tainer, Yin Shi, Zu Ye, Xiangdong Cheng

Faculty, Staff and Student Publications

Radiotherapy is one of the most important adjuvant treatment methods for gastric cancer (GC). However, radioresistance remains a major clinical obstacle. In this study, APOL2 is identified as a key player in promoting non-homologous end joining (NHEJ)-mediated double-strand break (DSB) repair and enhancing radioresistance in GC. Bioinformatics and clinical data revealed that high APOL2 expression is correlated with poor prognosis in GC patients. Functional experiments showed that APOL2 overexpression enhances genomic stability by accelerating DSB repair via the NHEJ pathway, while APOL2 knockout impairs repair capacity. Mechanistically, APOL2 binds to and stabilizes Ku80 by enhancing USP7-mediated deubiquitylation, thereby increasing Ku80 …

Brain-Wide Patterns Of Crf Receptor 1 Expression In The Transgenic Crfr1-Cre-2a-Tdtom Rat, Douglas S Engelke, Lierni Ugartemendia, Jonathan Tao, Shivakumar Rajamanikam, Fabricio H Do Monte, Nicholas J Justice

Faculty, Staff and Student Publications

The ability to manipulate neurons that express corticotropin-releasing factor (CRF) or its receptors has led to many new discoveries about how CRF signaling modulates stress-related behaviors in mice. However, similar advances in rats have been limited by the lack of reporter lines targeting CRF receptors, restricting access to behavioral paradigms that are more suitable for, or specific to, rats. Here, we provide an extensive neuroanatomical characterization of a recently generated CRFR1-Cre rat. We show that Cre and tdTomato are expressed in a pattern that closely aligns with previously reported CRFR1 expression patterns, as determined by in situ hybridization in rats …

Protein Kinase Giα Oxidation Negatively Regulates Antibody Production By B Cells, Hyun-Ju Cho, Rebecca L Charles, Oleksandra Prysyazhna, Sapna Arjun, Asvi A Francois, Kevin M Mcbride, Philip Eaton

Faculty, Staff and Student Publications

Endogenous oxidants induce a C42-dependent interprotein disulfide between the subunits of protein kinase G (PKG) Iα in cardiovascular tissues to control blood pressure and ventricular relaxation during diastole. PKGIα is expressed in other cell types, including those of the immune system, where the redox state of this kinase is likely to regulate other important physiological processes. The role of PKGIα oxidation in antibody production by B cells, which produce oxidants such as hydrogen peroxide during differentiation, was examined by comparing the immune response of oxidation-resistant C42S PKGIα knock-in (KI) mice to their wild type (WT) littermates.

Immunization with the 4 …

Sars-Cov-2 Mrna Vaccines Sensitize Tumours To Immune Checkpoint Blockade, Adam J Grippin, Christiano Marconi, Sage Copling, Nan Li, Chen Braun, Cole Woody, Elliana Young, Priti Gupta, Min Wang, Annette Wu, Seong Dong Jeong, Dhruvkumar Soni, Frances Weidert, Chao Xie, Eden Goldenberg, Andrew Kim, Chong Zhao, Anna Devries, Paul Castillo, Rishabh Lohray, Michael K Rooney, Benjamin R Schrank, Yifan Wang, Yifan Ma, Enoch Chang, Ramez Kouzy, Kyle Dyson, Jordan Jafarnia, Nina Nariman, Gregory Gladish, Jacob New, Ada Argueta, Diana Amaya, Nagheme Thomas, Andria Doty, Joe Chen, Nikhil Copling, Gabriel Alatrash, Julie Simon, Alicia Bea Davies, William Dennis, Richard Liang, Jeff Lewis, Xiong Wei, Waree Rinsurongkawong, Ara A Vaporciyan, Andrew Johns, D3code Team, Jack Lee, Ji-Hyun Lee, Ryan Sun, Padmanee Sharma, Hai Tran, Jianjun Zhang, Don L Gibbons, Jennifer Wargo, Betty Y S Kim, John V Heymach, Hector R Mendez-Gomez, Wen Jiang, Elias J Sayour, Steven H Lin

Faculty, Staff and Student Publications

Immune checkpoint inhibitors (ICIs) extend survival in many patients with cancer but are ineffective in patients without pre-existing immunity1–9. Although personalized mRNA cancer vaccines sensitize tumours to ICIs by directing immune attacks against preselected antigens, personalized vaccines are limited by complex and time-intensive manufacturing processes10–14. Here we show that mRNA vaccines targeting SARS-CoV-2 also sensitize tumours to ICIs. In preclinical models, SARS-CoV-2 mRNA vaccines led to a substantial increase in type I interferon, enabling innate immune cells to prime CD8+ T cells that target tumour-associated antigens. Concomitant ICI treatment is required for …