Tagless LysoIP for immunoaffinity enrichment of native lysosomes from clinical samples

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Abstract

Lysosomes are implicated in a wide spectrum of human diseases including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration and cancer. Profiling lysosomal content using tag-based lysosomal immunoprecipitation (LysoTagIP) in cell and animal models has substantially moved the field forward, but studying lysosomal dysfunction in human patients remains challenging. Here, we report the development of the ‘tagless LysoIP’ method, designed to enable the rapid enrichment of lysosomes, via immunoprecipitation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cell lines (e.g., induced pluripotent stem cell derived neurons). Isolated lysosomes were intact and suitable for subsequent multimodal omics analyses. To validate our approach, we applied the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells derived from fresh blood of healthy donors and patients with CLN3 disease, an autosomal recessive neurodegenerative LSD. Metabolic profiling of isolated lysosomes revealed massive accumulation of glycerophosphodiesters (GPDs) in patients’ lysosomes. Interestingly, a patient with a milder phenotype and genotype displayed lower accumulation of lysosomal GPDs, consistent with their potential role as disease biomarkers. Altogether, the tagless LysoIP provides a framework to study native lysosomes from patient samples, identify disease biomarkers, and discover human-relevant disease mechanisms.

Authors

Daniel Saarela, Pawel Lis, Sara Gomes, Raja S. Nirujogi, Wentao Dong, Eshaan S. Rawat, Sophie Glendinning, Karolina Zeneviciute, Enrico Bagnoli, Rotimi Fasimoye, Cindy Lin, Kwamina Nyame, Fanni A. Boros, Friederike Zunke, Frederic Lamoliatte, Sadik Elshani, Matthew Jaconelli, Judith J.M. Jans, Margriet A. Huisman, Christian Posern, Lena M. Westermann, Angela Schulz, Peter M. van Hasselt, Dario R. Alessi, Monther Abu-Remaileh, Esther M. Sammler

Targeted degradation of oncogenic KRAS^G12V triggers antitumor immunity in lung cancer models

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Abstract

KRAS is the most frequently mutated oncogene in lung adenocarcinoma, with G12C and G12V being the most predominant forms. Recent breakthroughs in KRASG12C inhibitors have transformed the clinical management of patients with G12C mutation and advanced our understanding of its function. However, little is known about the targeted disruption of KRASG12V, partly due to a lack of specific inhibitors. Here, we leverage the degradation tag (dTAG) system to develop a KRASG12V transgenic mouse model. We explore the therapeutic potential of KRASG12V degradation and characterize its impact on the tumor microenvironment (TME). Our study reveals that degrading KRASG12V abolishes lung and pancreatic tumors in mice and causes a robust inhibition of KRAS-regulated cancer intrinsic signaling. Importantly, targeted degradation of KRASG12V reprograms the TME towards a stimulatory milieu and drives antitumor immunity, elicited mainly by effector and cytotoxic CD8+ T cells. Our work provides important insights into the impact of degrading KRASG12V on both tumor progression and immune response, highlighting degraders as a powerful strategy for targeting KRAS mutant cancers.

Authors

Dezhi Li, Ke Geng, Yuan Hao, Jiajia Gu, Saurav Kumar, Annabel T. Olson, Christina C. Kuismi, Hye Mi Kim, Yuanwang Pan, Fiona Sherman, Asia M. Williams, Yiting Li, Fei Li, Ting Chen, Cassandra Thakurdin, Michela Ranieri, Mary Meynardie, Daniel S. Levin, Janaye Stephens, Alison Chafitz, Joy Chen, Mia S. Donald-Paladino, Jaylen M. Powell, Ze-Yan Zhang, Wei Chen, Magdalena Ploszaj, Han Han, Shengqing Gu, Tinghu Zhang, Baoli Hu, Benjamin A. Nacev, Medard Ernest Kaiza, Alice H. Berger, Xuerui Wang, Jing Li, Xuejiao Sun, Yang Liu, Xiaoyang Zhang, Tullia C. Bruno, Nathanael S. Gray, Behnam Nabet, Kwok-Kin Wong, Hua Zhang

A conserved human CD4⁺ T cell subset recognizing the mycobacterial adjuvant, trehalose monomycolate

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Abstract

Mycobacterium tuberculosis causes human tuberculosis. As mycobacteria are protected by thick lipid cell wall, humans have developed immune responses against diverse mycobacterial lipids. Most of these immunostimulatory lipids are known as adjuvants acting through innate immune receptors, such as C-type lectin receptors. Although a few mycobacterial lipid antigens activate unconventional T cells, antigenicity of most adjuvantic lipids are unknown. Here, we identified that trehalose monomycolate (TMM), an abundant mycobacterial adjuvant, activates human T cells bearing a unique ɑβTCR. This recognition was restricted by CD1b, a monomorphic antigen-presenting molecule conserved in primates but not mice. Single-cell TCR-RNA sequencing using newly established CD1b-TMM tetramers revealed that TMM-specific T cells are present as CD4+ effector memory T cells in the periphery of uninfected donors, but express IFNγ, TNF and anti-mycobacterial effectors upon TMM stimulation. TMM-specific T cells are detected in cord blood and PBMCs of non-BCG-vaccinated donors, but are expanded in active tuberculosis patients. A cryo-electron microscopy study of CD1b-TMM-TCR complexes revealed unique antigen recognition by conserved features of TCRs, positively-charged CDR3ɑ and long CDR3β regions. These results indicate that humans have a commonly-shared and pre-formed CD4+ T cell subset recognizing a typical mycobacterial adjuvant as an antigen. Furthermore, the dual role of TMM justifies reconsideration of the mechanism of action of adjuvants.

Authors

Yuki Sakai, Minori Asa, Mika Hirose, Wakana Kusuhara, Nagatoshi Fujiwara, Hiroto Tamashima, Takahiro Ikazaki, Shiori Oka, Kota Kuraba, Kentaro Tanaka, Takashi Yoshiyama, Masamichi Nagae, Yoshihiko Hoshino, Daisuke Motooka, Ildiko Van Rhijn, Xiuyuan Lu, Eri Ishikawa, D. Branch Moody, Takayuki Kato, Shinsuke Inuki, Go Hirai, Sho Yamasaki

CRISPR/Cas9 screens identify LIG1 as a sensitizer of PARP inhibitors in castration-resistant prostate cancer

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Abstract

PARP inhibitors (PARPi) have received regulatory approval for the treatment of several tumors, including prostate cancer (PCa), and demonstrate remarkable results in the treatment of castration-resistant prostate cancer (CRPC) patients characterized by defects in homologous recombination repair (HRR) genes. Preclinical studies showed that DNA repair genes (DRG) other than HRR genes may have therapeutic value in the context of PARPi. To this end, we performed multiple CRISPR/Cas9 screens in PCa cell lines using a custom sgRNA library targeting DRG combined with PARPi treatment. We identified LIG1, EME1, and FAAP24 losses as PARPi sensitizers and assessed their frequencies from 3 to 6% among CRPC patients. We showed that concomitant inactivation of LIG1 and PARP induced replication stress and DNA double-strand breaks, ultimately leading to apoptosis. This synthetic lethality (SL) is conserved across multiple tumor types (e.g., lung, breast, and colorectal), and its applicability might be extended to LIG1-functional tumors through a pharmacological combinatorial approach. Importantly, the sensitivity of LIG1-deficient cells to PARPi was confirmed in vivo. Altogether, our results argue for the relevance of determining the status of LIG1, and potentially other non-HRR DRG for CRPC patient stratification and provide evidence to expand their therapeutic options.

Authors

Giulia Fracassi, Francesca Lorenzin, Francesco Orlando, Ubaldo Gioia, Giacomo D'Amato, Arnau S. Casaramona, Thomas Cantore, Davide Prandi, Frédéric R. Santer, Helmut Klocker, Fabrizio d’Adda di Fagagna, Joaquin Mateo, Francesca Demichelis

Metalloprotease inhibitors regulate biliary progenitor cells through sDLK1 in organoid models of liver injury

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Abstract

Understanding cell fate regulation in the liver is necessary to advance cell therapies for hepatic disease. Liver progenitor cells (LPC) contribute to tissue regeneration after severe hepatic injury yet signals instructing progenitor cell dynamics and fate are largely unknown. The Tissue Inhibitor of Metalloproteinases, TIMP1 and TIMP3 control the sheddases ADAM10 and ADAM17, key for NOTCH activation. Here we uncover the role of the TIMP/ADAM/NOTCH/DLK1 axis in LPC maintenance and cholangiocyte specification. Combined TIMP1/TIMP3 loss in vivo caused abnormal portal triad stoichiometry accompanied by collagen deposits, dysregulated Notch signalling and increased soluble DLK1. The MIC1-1C3+CD133+CD26– biliary progenitor population was reduced following acute CCl4 or chronic DDC liver injury and in aged TIMP deficient livers. ScRNA-seq data interrogation and RNAscope identified portal mesenchymal cells co-expressing ADAM17/DLK1 as enzymatically equipped to process DLK1 and direct LPC differentiation. Specifically, TIMP deficient biliary fragment-derived organoids displayed increased propensity for cholangiocyte differentiation. ADAM17 inhibition reduced Sox9-mediated cholangiocyte differentiation, prolonging organoid growth and survival, whereas soluble DLK1-treated WT organoids triggered Sox9 expression and cholangiocyte specification in mouse and patient-derived liver organoids. Thus, metalloprotease inhibitors regulate instructive signals for biliary cell differentiation and LPC preservation within the portal niche, providing a new basis for cell therapy strategies.

Authors

Virginie Defamie, Kazeera Aliar, Soumili Sarkar, Foram Vyas, Ronak Shetty, Swami Reddy Narala, Hui Fang, Sanjay Saw, Pirashaanthy Tharmapalan, Otto Sanchez, Jennifer J. Knox, Paul D. Waterhouse, Rama Khokha

NEDD4L mediates intestinal epithelial cell ferroptosis to restrict inflammatory bowel diseases and colorectal tumorigenesis

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Abstract

Various factors play key roles in maintaining intestine homeostasis. Disruption of the balance may lead to intestinal inflammatory diseases (IBDs) and even colorectal cancer (CRC). Loss or gain of function of many key proteins can result in dysregulated intestinal homeostasis. Our research demonstrated that neural precursor cells expressed developmentally down-regulated 4-like protein, NEDD4L (NEDD4-2), a type of HECT family E3 ubiquitin ligase, played an important role in maintaining intestinal homeostasis. NEDD4L expression was significantly inhibited in intestinal epithelial cells (IECs) of patients with Crohn's disease (CD), ulcerative colitis (UC), and CRC. Global knockout of NEDD4L or its deficiency in IECs exacerbated dextran sulfate sodium (DSS)-/2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis and azoxymethane (AOM)/DSS-induced colorectal cancer. Mechanistically, NEDD4L deficiency in IECs inhibited the key ferroptosis regulator glutathione peroxidase 4 (GPX4) expression by reducing the protein expression of solute carrier family 3 member 2 (SLC3A2) without affecting its gene expression, ultimately promoting DSS-induced IEC ferroptosis. Importantly, ferroptosis inhibitors reduced the susceptibility of NEDD4L-deficient mice to colitis and colitis-associated colorectal cancer (CAC). Thus, NEDD4L was an important regulator in IEC ferroptosis, maintaining intestinal homeostasis, making it a potential clinical target for diagnosing and treating IBDs.

Authors

Jingjing Liang, Ning Wang, Yihan Yao, Yingmei Wang, Xiang An, Haofei Wang, Huan Liu, Yu Jiang, Hui Li, Xiaoqing Cheng, Jiaqi Xu, Xiaojing Liang, Jun Lou, Zengfeng Xin, Ting Zhang, Xiaojian Wang, Wenlong Lin

PINK1 is a target of T cell responses in Parkinson's disease

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Abstract

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder. While there is no curative treatment, the immune system's involvement with autoimmune T cells that recognize the protein alpha-synuclein (α-syn) in a subset of individuals suggests new areas for therapeutic strategies. As not all patients with PD have T cells specific for α-syn, we explored additional autoantigenic targets of T cells in PD. We generated 15-mer peptides spanning several PD-related proteins implicated in PD pathology, including Glucosylceramidase Beta 1 (GBA), Superoxide dismutase 1 (SOD1), PTEN Induced Kinase 1 (PINK1), Parkin RBR E3 Ubiquitin Protein Ligase (parkin), Oxoglutarate Dehydrogenase (OGDH), and Leucine Rich Repeat Kinase 2 (LRRK2). Cytokine production (IFNγ, IL-5, IL-10) against these proteins was measured using a fluorospot assay and PBMCs from patients with PD and age-matched healthy controls. We identified PINK1, a regulator of mitochondrial stability, as an autoantigen targeted by T cells, as well as its unique epitopes, and their HLA restriction. The PINK1-specific T cell reactivity revealed sex-based differences as it was predominantly found in male patients with PD, which may contribute to the heterogeneity of PD. Identifying and characterizing PINK1 and other autoinflammatory targets may lead to antigen-specific diagnostics, progression markers, and/or novel therapeutic strategies for PD.

Authors

Gregory P. Williams, Antoine Freuchet, Tanner Michaelis, April Frazier, Ngan K. Tran, João Rodrigues Lima-Junior, Elizabeth J. Phillips, Simon A. Mallal, Irene Litvan, Jennifer G. Goldman, Roy N. Alcalay, John Sidney, David Sulzer, Alessandro Sette, Cecilia S. Lindestam Arlehamn

Intestinal Cyp24a1 regulates vitamin D locally independent of systemic regulation by renal Cyp24a1 in mice

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Abstract

Vitamin D regulates mineral homeostasis. The most biologically active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D), is synthesized by CYP27B1 from 25-dihydroxyvitamin D (25D) and inactivated by CYP24A1. Human monogenic diseases and genome-wide association studies support a critical role for CYP24A1 in regulation of mineral homeostasis, but little is known about its tissue-specific effects. Here, we describe the responses of mice with inducible global deletion, kidney-specific, and intestine-specific deletion of Cyp24a1 to dietary calcium challenge and chronic kidney disease (CKD). Global and kidney-specific Cyp24a1 deletion caused similar syndromes of systemic vitamin D intoxication: elevated circulating 1,25D, 25D and fibroblast growth factor 23 (FGF23), activation of vitamin D target genes in the kidney and intestine, hypercalcemia, and suppressed parathyroid hormone (PTH). In contrast, mice with intestine-specific Cyp24a1 deletion demonstrated activation of vitamin D target genes exclusively in the intestine despite no changes in systemic vitamin D levels. In response to a high calcium diet, PTH was suppressed despite normal serum calcium. In mice with CKD, intestinal Cyp24a1 deletion decreased PTH and FGF23 without precipitating hypercalcemia. These results implicate kidney CYP24A1 in systemic vitamin D regulation while independent local effects of intestinal CYP24A1 could be targeted to treat secondary hyperparathyroidism in CKD.

Authors

Michaela A.A. Fuchs, Alexander Grabner, Melody Shi, Susan L. Murray, Emily J. Burke, Nejla Latic, Venkataramana Thiriveedi, Jatin Roper, Shintaro Ide, Koki Abe, Hiroki Kitai, Tomokazu Souma, Myles Wolf

Long COVID is associated with lower percentages of mature, cytotoxic NK cell phenotypes

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Abstract

Authors

Tasha Tsao, Amanda M. Buck, Lilian Grimbert, Brian H. LaFranchi, Belen Altamirano Poblano, Emily A. Fehrman, Thomas Dalhuisen, Priscilla Y. Hsue, J. Daniel Kelly, Jeffrey N. Martin, Steven G. Deeks, Peter W. Hunt, Michael J. Peluso, Oscar A. Aguilar, Timothy J. Henrich

Regulation of sarcomere formation and function in the healthy heart requires a titin intronic enhancer

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Abstract

Heterozygous truncating variants in the sarcomere protein titin (TTN) are the most common genetic cause of heart failure. To understand mechanisms that regulate abundant cardiomyocyte TTN expression we characterized highly conserved intron 1 sequences that exhibited dynamic changes in chromatin accessibility during differentiation of human cardiomyocytes from induced pluripotent stem cells (hiPSC-CMs). Homozygous deletion of these sequences in mice caused embryonic lethality while heterozygous mice demonstrated allele-specific reduction in Ttn expression. A 296 bp fragment of this element, denoted E1, was sufficient to drive expression of a reporter gene in hiPSC-CMs. Deletion of E1 downregulated TTN expression, impaired sarcomerogenesis, and decreased contractility in hiPSC-CMs. Site-directed mutagenesis of predicted NKX2-5- and MEF2-binding sites within E1 abolished its transcriptional activity. Embryonic mice expressing E1 reporter gene constructs validated in vivo cardiac-specific activity of E1 and the requirement for NKX2-5 and MEF2 binding sequences. Moreover, isogenic hiPSC-CMs containing a rare E1 variant in the predicted MEF2 binding motif that was identified in a patient with unexplained DCM showed reduced TTN expression. Together these discoveries define an essential, functional enhancer that regulates TTN expression. Manipulation of this element may advance therapeutic strategies to treat DCM caused by TTN haploinsufficiency.

Authors

Yuri Kim, Seong Won Kim, David Saul, Meraj Neyazi, Manuel Schmid, Hiroko Wakimoto, Neil Slaven, Joshua H. Lee, Olivia G. Layton, Lauren K. Wasson, Justin H. Letendre, Feng Xiao, Jourdan K. Ewoldt, Konstantinos Gkatzis, Peter Sommer, Bénédicte Gobert, Nicolas Wiest-Daesslé, Quentin McAfee, Nandita Singhal, Mingyue Lun, Joshua M. Gorham, Zoltan Arany, Arun Sharma, Christopher N. Toepfer, Gavin Y. Oudit, William T. Pu, Diane E. Dickel, Len A. Pennacchio, Axel Visel, Christopher S. Chen, J.G. Seidman, Christine E. Seidman