IFNγ-driven skewing towards Th1 over Th17 differentiation underlies CRS and neutropenia in CAR-T therapy

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Abstract

CAR-T therapy has led to significant improvements in patient survival. However, a subset of patients experience high-grade toxicities, including cytokine release syndrome (CRS) and immune cell-associated hematological toxicity (ICAHT). We utilized IL-2Rα knockout mice to model toxicities with elevated levels of IL6, IFNγ, and TNFα and increased M1-like macrophages. Onset of CRS was accompanied by a reduction in peripheral blood neutrophils due to disruption of bone marrow neutrophil homeostasis characterized by an increase in apoptotic neutrophils and a decrease in proliferative and mature neutrophils. Both non-tumor-bearing and Eμ-ALL tumor-bearing mice recapitulated the co-occurrence of CRS and neutropenia. IFNγ-blockade alleviated CRS and neutropenia without affecting CAR-T efficacy. Mechanistically, a Th1-Th17 imbalance was observed to drive co-occurrence of CRS and neutropenia in an IFNγ-dependent manner leading to decreased IL-17A and G-CSF, neutrophil production, and neutrophil survival. In patients, we observed an increase in the IFNγ-to-IL-17A ratio in the peripheral blood during high-grade CRS and neutropenia. We have uncovered a biological basis for ICAHT and provide support for the use of IFNγ-blockade to reduce both CRS and neutropenia.

Authors

Payal Goala, Yongliang Zhang, Nolan J. Beatty, Allan Pavy, Shannon L. McSain, Cooper J. Sailer, Muhammad Junaid Tariq, Showkat Hamid, Eduardo Cortes Gomez, Jianmin Wang, Duna Massillon, Maxwell Ilecki, Justin C. Boucher, Constanza Savid-Frontera, Sae Bom Lee, Hiroshi Kotani, Meredith L. Stone, Michael D. Jain, Marco L. Davila

Lnk Deficiency Enhances Translesion Synthesis to Alleviate Replication Stress and Promote Hematopoietic Stem Cell Fitness

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Abstract

The adaptor protein LNK/SH2B3 negatively regulates hematopoietic stem cell (HSC) homeostasis. Lnk-deficient mice show marked expansion of HSCs without premature exhaustion. Lnk deficiency largely restores HSC function in Fanconi Anemia (FA) mouse models and primary FA patient cells, albeit protective mechanisms remain enigmatic. Here, we uncover a novel role for LNK in regulating translesion synthesis (TLS) during HSC replication. Lnk deficiency reduced replication stress-associated DNA damage, particularly in the FA background. Lnk deficiency suppressed single-strand DNA breaks, while enhancing replication fork restart in FA-deficient HSCs. Diminished replication-associated damage in Lnk-deficient HSCs occurred commensurate with reduced ATR-p53 checkpoint activation that is linked to HSC attrition. Notably, Lnk deficiency ameliorated HSC attrition in FA mice without exacerbating carcinogenesis during ageing. Moreover, we demonstrated that enhanced HSC fitness from Lnk deficiency was associated with increased TLS activity via REV1 and, to a lesser extent, TLS polymerase eta. TLS polymerases are specialized to execute DNA replication in the presence of lesions or natural replication fork barriers that stall replicative polymerases. Our findings implicate elevated use of these specialized DNA polymerases as critical to the enhanced HSC function imparted by Lnk deficiency, which has important ramifications for stem cell therapy and regenerative medicine in general.

Authors

Brijendra Singh, Md Akram Hossain, Xiao Hua Liang, Jeremie Fages, Carlo Salas Salinas, Roger A. Greenberg, Wei Tong

Ciliopathy-related B9 protein complex regulates ciliary axonemal microtubule post-translational modifications and initiation of ciliogenesis

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Abstract

Ciliary dysfunction results in multi-organ involved developmental diseases, collectively known as ciliopathies. The B9D1-B9D2-MKS1 protein complex maintains the gatekeeper function at the ciliary transition zone (TZ). However, the function of B9 proteins and the mechanisms underlying why different variants in the same B9 gene cause different ciliopathies are not fully understood. Here, we investigated the function of B9 proteins and revealed two critical functions. First, the B9 complex interacted with and anchored TMEM67 to the TZ membrane. Disruption of the B9-TMEM67 complex reduced posttranslational-modifications of axonemal microtubules due to deregulation of tubulin-modifying enzymes within cilia. Second, B9 proteins localized to centrioles prior to ciliogenesis, where they facilitated the initiation of ciliogenesis. Finally, we identified B9D2 variants in a cohort of patients with Joubert syndrome (JBTS). Consistent with the dual functions, we found that the JBTS-associated B9D2 variants primarily affected axonemal microtubule modifications without disrupting ciliogenesis, whereas the Meckel syndrome (MKS)-associated B9D2 variant disrupted both ciliogenesis and axonemal microtubule modifications. Thus, besides its role as a gatekeeper for ciliary membrane proteins, the B9 complex also controls axonemal microtubule post-translational modifications and early stages of ciliogenesis, providing insights into the distinct pathologies arising from different variants of the same gene.

Authors

Ruida He, Yan Li, Minjun Jin, Huike Jiao, Yue Shen, Qize Han, Xilang Pan, Suning Wang, Zaisheng Lin, Jingshi Li, Chao Lu, Dan Meng, Zongfu Cao, Qing Shang, Nan Lv, Kai Wan, Huafang Gao, Xu Ma, Haiyan Yin, Haishuang Chang, Liang Wang, Minna Luo, Junmin Pan, Chengtian Zhao, Muqing Cao

Molecular glue degrader function of SPOP enhances STING-dependent immunotherapy efficacy in melanoma models

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Abstract

The E3 ligase SPOP plays a context-dependent role in cancer by targeting specific cellular proteins for degradation, thereby influencing cell behavior. However, its role in tumor immunity remains largely unexplored. In this study, we revealed that SPOP targeted the innate immune sensor STING for degradation in a CK1γ phosphorylation-dependent manner to promote melanoma growth. Stabilization of STING by escaping SPOP-mediated degradation enhanced anti-tumor immunity by increasing IFNβ production and ISG expression. Notably, small-molecule SPOP inhibitors not only blocked STING recognition by SPOP, but also acted as molecular glues, redirecting SPOP to target neo-substrates such as CBX4 for degradation. This CBX4 degradation led to increased DNA damage, which in turn activated STING and amplified innate immune responses. In a xenografted melanoma B16 tumor model, single-cell RNA-seq analysis demonstrated that SPOP inhibition induced the infiltration of immune cells associated with anti-PD1 responses. Consequently, SPOP inhibitors synergized with immune checkpoint blockade to suppress B16 tumor growth in syngeneic murine models and enhanced the efficacy of CD19-CAR-T therapy. Our findings highlight a molecular glue degrader property of SPOP inhibitors, with potential implications for other E3 ligase-targeting small molecules designed to disrupt protein-protein interactions.

Authors

Zhichuan Zhu, Xin Zhou, Max Xu, Jianfeng Chen, Kevin C. Robertson, Gatphan N. Atassi, Mark G. Woodcock, Allie C. Mills, Laura E. Herring, Gianpietro Dotti, Pengda Liu

Secretory kinase FAM20C triggers adipocyte dysfunction inciting insulin resistance and inflammation in obesity

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Abstract

Obesity is a major driver of type 2 diabetes (T2D) and related metabolic disorders, characterized by chronic inflammation and adipocyte dysfunction. However, the molecular triggers initiating these processes remain poorly understood. We identify FAM20C, a serine/threonine kinase, as an early obesity-induced mediator of adipocyte dysfunction. Fam20c expression is substantially upregulated in adipocytes in response to obesity, correlating with a proinflammatory transcriptional signature. Forced expression of Fam20c in adipocytes promotes robust upregulation of proinflammatory cytokines and induces insulin resistance that is dependent on its kinase activity. Conversely, deletion of adipocyte Fam20c after established obesity and hyperglycemia improves glucose tolerance, augments insulin sensitivity, and reduces visceral adiposity, without altering body weight. Phosphoproteomic studies reveal that FAM20C regulates phosphorylation of intracellular and secreted proteins, modulating pathways critical to inflammation, metabolism, and extracellular matrix remodeling. We identify FAM20C-dependent substrates, such as CNPY4, whose phosphorylation contributes to proinflammatory adipocyte signaling. Of translational relevance, we show that in humans visceral adipose FAM20C expression positively correlates with insulin resistance. Our findings establish FAM20C as an early regulator of obesity-induced adipocyte dysfunction and systemic metabolic impairment. Our studies provide proof of concept that inhibition of FAM20C may serve as a potential therapy for T2D by restoring adipocyte health.

Authors

Ankit Gilani, Benjamin D. Stein, Anne Hoffmann, Renan Pereira de Lima, Elizabeth E. Ha, Edwin A. Homan, Lunkun Ma, Alfonso Rubio-Navarro, Tint Tha Ra Wun, Gabriel Jose Ayala Carrascal, Bhavneet Bhinder, Adhideb Ghosh, Falko J. Noé, Olivier Elemento, Christian Wolfrum, Matthias Blüher, James C. Lo

Tissue-specific anti-tumor NK cell subsets identified in colorectal cancer liver metastases express candidate therapeutic targets

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Abstract

Liver metastases are relatively resistant to checkpoint blockade immunotherapy. The hepatic tissue has distinctive features including high numbers of NK cells. It was therefore important to conduct in depth single-cell analysis of NK cells in colorectal cancer liver metastases (CRLMs) with the effort to dissect their diversity and to identify candidate therapeutic targets. By combining unbiased single-cell transcriptomic with multiparametric flow cytometry analysis, we identified an abundant family of intrahepatic CD56Bright NK cells in CRLMs endowed with anti-tumor functions resulting from specific transcriptional liver programs. Intrahepatic CD56Bright and CD56Dim NK lymphocytes expressed unique transcription factors (IRF8, TOX2), high level of chemokines, and targetable immune checkpoints (ICs), including CXCR4 and the IL-1 receptor family member IL-1R8. CXCR4 pharmacological blocking and an anti-IL-1R8 mAb enhanced the effector function of CRLM NK cells. Targeting the diversity of liver NK cells and their distinct immune-checkpoint repertoires is key to optimize the current immune-therapy protocols in CRLM.

Authors

Joanna Mikulak, Domenico Supino, Paolo Marzano, Sara Terzoli, Roberta Carriero, Valentina Cazzetta, Rocco Piazza, Elena Bruni, Paolo Kunderfranco, Alessia Donato, Sarah Natalia Mapelli, Roberto Garuti, Silvia Carnevale, Francesco Scavello, Elena Magrini, Jelena Zeleznjak, Clelia Peano, Matteo Donadon, Guido Costa, Guido Torzilli, Alberto Mantovani, Cecilia Garlanda, Domenico Mavilio

T-cell acute lymphoblastic leukemia exploits a neural proinflammatory pathway to colonize the meninges

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Abstract

Infiltration of T-cell acute lymphoblastic leukemia (T-ALL) into the meninges worsens prognosis, underscoring the need to understand mechanisms driving meningeal involvement. Here, we show that T-ALL cells expressing CXCR3 exploit normal T-cell function to infiltrate the inflamed meninges. CXCR3 deletion hampered disease progression and extramedullary dissemination by reducing leukemic cell proliferation and migration. Conversely, forced expression of CXCR3 facilitated T-ALL trafficking to the meninges. We identified the ubiquitin-specific protease 7 as a key regulator of CXCR3 protein stability in T-ALL. Furthermore, we discovered elevated levels of CXCL10, a CXCR3 ligand, in the cerebrospinal fluid from T-ALL patients and leukemia-bearing mice. Our studies demonstrate that meningeal stromal cells, specifically pericytes and fibroblasts, induce CXCL10 expression in response to leukemia, and that loss of CXCL10 attenuated T-ALL influx into the meninges. Moreover, we report that leukemia-derived proinflammatory cytokines, TNFα, IL27 and IFNγ, induced CXCL10 in the meningeal stroma. Pharmacological inhibition or deletion of CXCR3 or CXCL10 reduced T-ALL cell migration and adhesion to meningeal stromal cells. Finally, we reveal that CXCR3 and CXCL10 upregulated VLA-4/VCAM-1 signaling, promoting cell-cell adhesion and thus T-ALL retention in the meninges. Our findings highlight the pivotal role of CXCR3-CXCL10 signaling in T-ALL progression and meningeal colonization.

Authors

Nitesh D. Sharma, Esra'a Keewan, Wojciech Ornatowski, Silpita Paul, Monique Nysus, Christopher C. Barnett, Julie Wolfson, Quiteria Jacquez, Bianca L. Myers, Huining Kang, Katherine E. Zychowski, Stuart S. Winter, Mignon L. Loh, Stephen P. Hunger, Eliseo F. Castillo, Tom Taghon, Christina Halsey, Tou Yia Vue, Nicholas Jones, Panagiotis Ntziachristos, Ksenia Matlawska-Wasowska

The hematopoietic stem cell MYB enhancer is essential and recurrently amplified during T-cell leukemogenesis

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Abstract

There is an urgent need to find targeted agents for T-cell acute lymphoblastic leukemia (T-ALL). NOTCH1 is the most frequently mutated oncogene in T-ALL, but clinical trials showed that pan-Notch inhibitors caused dose-limiting toxicities. Thus, we shifted our focus to ETS1, which is one of the transcription factors that most frequently co-bind Notch-occupied regulatory elements in the T-ALL context. To identify the most essential enhancers, we performed a genome-wide CRISPR interference screen of the strongest ETS1-dependent regulatory elements. The #1-ranked element is located in an intron of AHI1 that interacts with the MYB promoter and is amplified with MYB in ~8.5% of T-ALL patients. Using mouse models, we showed that this enhancer promotes self-renewal of hematopoietic stem cells and T-cell leukemogenesis, maintains early T-cell precursors, and restrains myeloid expansion with aging. We named this enhancer the hematopoietic stem cell MYB enhancer (H-Me). The H-Me shows limited activity and function in committed T-cell progenitors but is accessed during leukemogenesis. In one T-ALL context, ETS1 binds the ETS motif in the H-Me to recruit cBAF to promote chromatin accessibility and activation. ETS1 or cBAF degraders impaired H-Me function. Thus, we identified a targetable stem cell element that is co-opted for T-cell transformation.

Authors

Carea Mullin, Karena Lin, Elizabeth Choe, Cher Sha, Zeel Shukla, Koral Campbell, Anna C. McCarter, Annie Wang, Jannaldo Nieves-Salva, Sarah Khan, Theresa M. Keeley, Shannon Liang, Qing Wang, Ashley F. Melnick, Pearl Evans, Alexander C. Monovich, Ashwin Iyer, Rohan Kodgule, Yamei Deng, Felipe da Veiga Leprevost, Kelly R. Barnett, Petri Pölönen, Rami Khoriaty, Daniel Savic, David T. Teachey, Charles G. Mullighan, Marcin Cieslik, Alexey I. Nesvizhskii, Linda C. Samuelson, Morgan Jones, Qing Li, Russell J.H. Ryan, Mark Y. Chiang

Distinct human small intestinal microbiome communities underlie visceral hypersensitivity in a humanized mouse model

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Abstract

Authors

Isin Y. Comba, Tijs Louwies, Ruben A. Mars, Yang Xiao, Prabhjot Kaur Sekhon, Brian S. Edwards, Adam Willits, Robin R. Shields-Cutler, Shreya Bellampalli, Arnaldo Mercado-Perez, Dennis R. Tienter, Lisa M. Till, David R. Linden, Gianrico Farrugia, Arthur Beyder, Kristen M. Smith-Edwards, Purna C. Kashyap

Spinal α2δ-1 induces GluA3 degradation to regulate assembly of calcium-permeable AMPA receptors and pain hypersensitivity

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Abstract

The increased prevalence of GluA2-lacking, Ca2+-permeable AMPA receptors (CP-AMPARs) at spinal cord sensory synapses amplifies nociceptive transmission and maintains chronic neuropathic pain. Nerve injury–induced upregulation of α2δ-1 disrupts the assembly of GluA1/GluA2 heteromers, favoring the synaptic incorporation of GluA1 homotetramers in the spinal dorsal horn. Although GluA1-GluA3 subunits are broadly expressed, whether α2δ-1 regulates GluA3-containing AMPARs remains unknown. Here, we unexpectedly found that coexpression with α2δ-1—but not α2δ-2 or α2δ-3—diminished GluA3 AMPAR currents and protein levels, an effect blocked by pregabalin, an α2δ-1 C-terminus peptide, or proteasome inhibition. Both nerve injury and α2δ-1 overexpression reduced protein levels of GluA3 and GluA2/GluA3 heteromers in the spinal cord. Furthermore, α2δ-1 coexpression or nerve injury increased GluA3 ubiquitination, with Lys-861 at the C terminus of GluA3 identified as a key ubiquitination site mediating α2δ-1–induced GluA3 degradation. Additionally, intrathecal delivery of the Gria3 gene reversed nerve injury–induced nociceptive hypersensitivity and synaptic CP-AMPARs by restoring protein levels of GluA3 and GluA2/GluA3 heteromers in the spinal cord. These findings reveal that α2δ-1 promotes GluA1 homotetramer assembly and synaptic CP-AMPAR expression by driving ubiquitin-proteasomal degradation of GluA3, providing insights into the molecular mechanisms of neuropathic pain and the therapeutic actions of gabapentinoids.

Authors

Meng-Hua Zhou, Shao-Rui Chen, Daozhong Jin, Yuying Huang, Hong Chen, Guanxing Chen, Jiusheng Yan, Hui-Lin Pan