Inhibition of phosphodiesterase 4D suppresses mTORC1 signaling and pancreatic cancer growth
Jeong et al. found that phosphodiesterase 4D plays a critical role in regulating Raptor Ser791 phosphorylation and mTORC1 activity in pancreatic cancer. The cover image is an artistic depiction of mTORC1 at the lysosome. Image credit: Jose Cabrera and Tshering Lama-Sherpa.
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Research Articles
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Abstract
Mechanical, thermal, and chemical pain sensation is conveyed by primary nociceptors, a subset of sensory afferent neurons. The intracellular regulation of the primary nociceptive signal is an area of active study. We report here the discovery of a Gβ5-dependent regulatory pathway within mechanical nociceptors that restrains antinociceptive input from metabotropic GABA-B receptors. In mice with conditional knockout (cKO) of the gene that encodes Gβ5 (Gnb5) targeted to peripheral sensory neurons, we demonstrate the impairment of mechanical, thermal, and chemical nociception. We further report the specific loss of mechanical nociception in Rgs7-Cre+/– Gnb5fl/fl mice but not in Rgs9-Cre+/– Gnb5fl/fl mice, suggesting that Gβ5 might specifically regulate mechanical pain in regulator of G protein signaling 7–positive (Rgs7+) cells. Additionally, Gβ5-dependent and Rgs7-associated mechanical nociception is dependent upon GABA-B receptor signaling since both were abolished by treatment with a GABA-B receptor antagonist and since cKO of Gβ5 from sensory cells or from Rgs7+ cells potentiated the analgesic effects of GABA-B agonists. Following activation by the G protein–coupled receptor Mrgprd agonist β-alanine, enhanced sensitivity to inhibition by baclofen was observed in primary cultures of Rgs7+ sensory neurons harvested from Rgs7-Cre+/– Gnb5fl/fl mice. Taken together, these results suggest that the targeted inhibition of Gβ5 function in Rgs7+ sensory neurons might provide specific relief for mechanical allodynia, including that contributing to chronic neuropathic pain, without reliance on exogenous opioids.
Authors
Mritunjay Pandey, Jian-Hua Zhang, Poorni R. Adikaram, Claire Kittock, Nicole Lue, Adam Awe, Katherine Degner, Nirmal Jacob, Jenna Staples, Rachel Thomas, Allison B. Kohnen, Sundar Ganesan, Juraj Kabat, Ching-Kang Chen, William F. Simonds
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Glioblastomas (GBM) are aggressive tumors that lack effective treatments. Here, we show that the Rho family guanine nucleotide exchange factor Syx promotes GBM cell growth both in vitro and in orthotopic xenografts derived from patients with GBM. Growth defects upon Syx depletion are attributed to prolonged mitosis, increased DNA damage, G2/M cell cycle arrest, and cell apoptosis, mediated by altered mRNA and protein expression of various cell cycle regulators. These effects are phenocopied by depletion of the Rho downstream effector Dia1 and are due, at least in part, to increased phosphorylation, cytoplasmic retention, and reduced activity of the YAP/TAZ transcriptional coactivators. Furthermore, targeting Syx signaling cooperates with radiation treatment and temozolomide (TMZ) to decrease viability in GBM cells, irrespective of their inherent response to TMZ. The data indicate that a Syx-RhoA-Dia1-YAP/TAZ signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in GBM and argue for its targeting for cancer treatment.
Authors
Wan-Hsin Lin, Ryan W. Feathers, Lisa M. Cooper, Laura J. Lewis-Tuffin, Jiaxiang Chen, Jann N. Sarkaria, Panos Z. Anastasiadis
×Abstract
The mammalian target of rapamycin complex 1 (mTORC1) senses multiple upstream stimuli to orchestrate anabolic and catabolic events that regulate cell growth and metabolism. Hyperactivation of mTORC1 signaling is observed in multiple human diseases; thus, pathways that suppress mTORC1 signaling may help to identify new therapeutic targets. Here, we report that phosphodiesterase 4D (PDE4D) promotes pancreatic cancer tumor growth by increasing mTORC1 signaling. GPCRs paired to Gαs proteins activate adenylyl cyclase, which in turn elevates levels of 3′,5′-cyclic adenosine monophosphate (cAMP), whereas PDEs catalyze the hydrolysis of cAMP to 5′-AMP. PDE4D forms a complex with mTORC1 and is required for mTORC1 lysosomal localization and activation. Inhibition of PDE4D and the elevation of cAMP levels block mTORC1 signaling via Raptor phosphorylation. Moreover, pancreatic cancer exhibits an upregulation of PDE4D expression, and high PDE4D levels predict the poor overall survival of patients with pancreatic cancer. Importantly, FDA-approved PDE4 inhibitors repress pancreatic cancer cell tumor growth in vivo by suppressing mTORC1 signaling. Our results identify PDE4D as an important activator of mTORC1 and suggest that targeting PDE4 with FDA-approved inhibitors may be beneficial for the treatment of human diseases with hyperactivated mTORC1 signaling.
Authors
Mi-Hyeon Jeong, Greg Urquhart, Cheryl Lewis, Zhikai Chi, Jenna L. Jewell
×Abstract
Pericytes are multipotent mesenchymal precursor cells that demonstrate tissue-specific properties. In this study, by comparing human adipose tissue– and periosteum-derived pericyte microarrays, we identified T cell lymphoma invasion and metastasis 1 (TIAM1) as a key regulator of cell morphology and differentiation decisions. TIAM1 represented a tissue-specific determinant between predispositions for adipocytic versus osteoblastic differentiation in human adipose tissue–derived pericytes. TIAM1 overexpression promoted an adipogenic phenotype, whereas its downregulation amplified osteogenic differentiation. These results were replicated in vivo, in which TIAM1 misexpression altered bone or adipose tissue generation in an intramuscular xenograft animal model. Changes in pericyte differentiation potential induced by TIAM1 misexpression correlated with actin organization and altered cytoskeletal morphology. Small molecule inhibitors of either small GTPase Rac1 or RhoA/ROCK signaling reversed TIAM1-induced morphology and differentiation in pericytes. In summary, our results demonstrate that TIAM1 regulates the cellular morphology and differentiation potential of human pericytes, representing a molecular switch between osteogenic and adipogenic cell fates.
Authors
Ginny Ching-Yun Hsu, Yiyun Wang, Amy Z. Lu, Mario A. Gomez-Salazar, Jiajia Xu, Dongqing Li, Carolyn Meyers, Stefano Negri, Sintawat Wangsiricharoen, Kristen Broderick, Bruno Peault, Carol Morris, Aaron W. James
×Abstract
Intratumoral heterogeneity is a defining hallmark of glioblastoma, driving drug resistance and ultimately recurrence. Many somatic drivers of microenvironmental change have been shown to affect this heterogeneity and, ultimately, the treatment response. However, little is known about how germline mutations affect the tumoral microenvironment. Here, we find that the single-nucleotide polymorphism (SNP) rs755622 in the promoter of the cytokine macrophage migration inhibitory factor (MIF) is associated with increased leukocyte infiltration in glioblastoma. Furthermore, we identified an association between rs755622 and lactotransferrin expression, which could also be used as a biomarker for immune-infiltrated tumors. These findings demonstrate that a germline SNP in the promoter region of MIF may affect the immune microenvironment and further reveal a link between lactotransferrin and immune activation.
Authors
Tyler J. Alban, Matthew M. Grabowski, Balint Otvos, Defne Bayik, Wesley Wang, Ajay Zalavadia, Vlad Makarov, Katie Troike, Mary McGraw, Anja Rabljenovic, Adam Lauko, Chase Neumann, Gustavo Roversi, Kristin A. Waite, Gino Cioffi, Nirav Patil, Thuy T. Tran, Kathleen McCortney, Alicia Steffens, C. Marcela Diaz, J. Mark Brown, Kathleen M. Egan, Craig M. Horbinski, Jill S. Barnholtz-Sloan, Prajwal Rajappa, Michael A. Vogelbaum, Richard Bucala, Timothy A. Chan, Manmeet S. Ahluwalia, Justin D. Lathia
×Abstract
Excessive activation of cardiac fibroblasts (CFs) in response to injury provokes cardiac fibrosis, stiffness, and failure. The local mediators counterregulating this response remain unclear. Exogenous C-type natriuretic peptide (CNP) exerts antifibrotic effects in preclinical models. To unravel the role of the endogenous hormone, we generated mice with fibroblast-restricted deletion (KO) of guanylyl cyclase-B (GC-B), the cGMP-synthesizing CNP receptor. CNP activated GC-B/cGMP signaling in human and murine CFs, preventing proliferative and promigratory effects of angiotensin II (Ang II) and TGF-β. Fibroblast-specific GC-B–KO mice showed enhanced fibrosis in response to Ang II infusions. Moreover, after 2 weeks of mild pressure overload induced by transverse aortic constriction (TAC), such KO mice had augmented cardiac fibrosis and hypertrophy, together with systolic and diastolic contractile dysfunction. This was associated with increased expression of the profibrotic genes encoding collagen I, III, and periostin. Notably, such responses to Ang II and TAC were greater in female as compared with male KO mice. Enhanced Ang II–induced CNP expression in female hearts and augmented GC-B expression and activity in female CFs may contribute to this sex disparity. The results show that paracrine CNP signaling in CFs has antifibrotic and antihypertrophic effects. The CNP/GC-B/cGMP pathway might be a target for therapies combating pathological cardiac remodeling.
Authors
Franziska Werner, Estefania Prentki Santos, Konstanze Michel, Hanna Schrader, Katharina Völker, Tamara Potapenko, Lisa Krebes, Marco Abeßer, Dorothe Möllmann, Martin Schlattjan, Hannes Schmidt, Boris V. Skryabin, Katarina Špiranec Spes, Kai Schuh, Christopher P. Denton, Hideo A. Baba, Michaela Kuhn
×Abstract
Thymic stromal lymphopoietin (TSLP) overexpression is widely associated with atopy. However, TSLP is expressed in normal barrier organs, suggesting a homeostatic function. To determine the function of TSLP in barrier sites, we investigated the impact of endogenous TSLP signaling on the homeostatic expansion of CD4+ T cells in adult mice. Surprisingly, incoming CD4+ T cells induced lethal colitis in adult Rag1-knockout animals that lacked the TSLP receptor (Rag1KOTslprKO). Endogenous TSLP signaling was required for reduced CD4+ T cell proliferation, Treg differentiation, and homeostatic cytokine production. CD4+ T cell expansion in Rag1KOTslprKO mice was dependent on the gut microbiome. The lethal colitis was rescued by parabiosis between Rag1KOTslprKO and Rag1KO animals and wild-type dendritic cells (DCs) suppressed CD4+ T cell–induced colitis in Rag1KOTslprKO mice. A compromised T cell tolerance was noted in TslprKO adult colon, which was exacerbated by anti–PD-1 and anti–CTLA-4 therapy. These results reveal a critical peripheral tolerance axis between TSLP and DCs in the colon that blocks CD4+ T cell activation against the commensal gut microbiome.
Authors
Jonathan L. Messerschmidt, Marjan Azin, Kaitlin E. Dempsey, Shadmehr Demehri
×Abstract
Cutaneous T cell lymphoma (CTCL) is a disfiguring and incurable disease characterized by skin-homing malignant T cells surrounded by immune cells that promote CTCL growth through an immunosuppressive tumor microenvironment (TME). Preliminary data from our phase I clinical trial of anti–programmed cell death ligand 1 (anti–PD-L1) combined with lenalidomide in patients with relapsed/refractory CTCL demonstrated promising clinical efficacy. In the current study, we analyzed the CTCL TME, which revealed a predominant PD-1+ M2-like tumor-associated macrophage (TAM) subtype with upregulated NF-κB and JAK/STAT signaling pathways and an aberrant cytokine and chemokine profile. Our in vitro studies investigated the effects of anti–PD-L1 and lenalidomide on PD-1+ M2-like TAMs. The combinatorial treatment synergistically induced functional transformation of PD-1+ M2-like TAMs toward a proinflammatory M1-like phenotype that gained phagocytic activity upon NF-κB and JAK/STAT inhibition, altered their migration through chemokine receptor alterations, and stimulated effector T cell proliferation. Lenalidomide was more effective than anti–PD-L1 in downregulation of the immunosuppressive IL-10, leading to decreased expression of both PD-1 and PD-L1. Overall, PD-1+ M2-like TAMs play an immunosuppressive role in CTCL. Anti–PD-L1 combined with lenalidomide provides a therapeutic strategy to enhance antitumor immunity by targeting PD-1+ M2-like TAMs in the CTCL TME.
Authors
Zhen Han, Xiwei Wu, Hanjun Qin, Yate-Ching Yuan, Daniel Schmolze, Chingyu Su, Jasmine Zain, Lilach Moyal, Emmilia Hodak, James F. Sanchez, Peter P. Lee, Mingye Feng, Steven T. Rosen, Christiane Querfeld
×Abstract
Osteosarcoma (OS) is the most common primary bone tumor of childhood. Approximately 20%–30% of OSs carry amplification of chromosome 8q24, which harbors the oncogene c-MYC and correlates with a poor prognosis. To understand the mechanisms that underlie the ability of MYC to alter both the tumor and its surrounding tumor microenvironment (TME), we generated and molecularly characterized an osteoblast-specific Cre-Lox-Stop-Lox-c-MycT58A p53fl/+ knockin genetically engineered mouse model (GEMM). Phenotypically, the Myc-knockin GEMM had rapid tumor development with a high incidence of metastasis. MYC-dependent gene signatures in our murine model demonstrated significant homology to the human hyperactivated MYC OS. We established that hyperactivation of MYC led to an immune-depleted TME in OS demonstrated by the reduced number of leukocytes, particularly macrophages. MYC hyperactivation led to the downregulation of macrophage colony-stimulating factor 1, through increased microRNA 17/20a expression, causing a reduction of macrophage population in the TME of OS. Furthermore, we developed cell lines from the GEMM tumors, including a degradation tag–MYC model system, which validated our MYC-dependent findings both in vitro and in vivo. Our studies utilized innovative and clinically relevant models to identify a potentially novel molecular mechanism through which MYC regulates the profile and function of the OS immune landscape.
Authors
Bikesh K. Nirala, Tajhal D. Patel, Lyazat Kurenbekova, Ryan Shuck, Atreyi Dasgupta, Nino Rainusso, Cristian Coarfa, Jason T. Yustein
×Abstract
B cells contribute to multiple aspects of autoimmune disorders, and B cell–targeting therapies, including B cell depletion, have been proven to be efficacious in treatment of multiple autoimmune diseases. However, the development of novel therapies targeting B cells with higher efficacy and a nondepleting mechanism of action is highly desirable. Here we describe a nondepleting, high-affinity anti–human CD19 antibody LY3541860 that exhibits potent B cell inhibitory activities. LY3541860 inhibits B cell activation, proliferation, and differentiation of primary human B cells with high potency. LY3541860 also inhibits human B cell activities in vivo in humanized mice. Similarly, our potent anti-mCD19 antibody also demonstrates improved efficacy over CD20 B cell depletion therapy in multiple B cell–dependent autoimmune disease models. Our data indicate that anti-CD19 antibody is a highly potent B cell inhibitor that may have potential to demonstrate improved efficacy over currently available B cell–targeting therapies in treatment of autoimmune conditions without causing B cell depletion.
Authors
Jeffrey S. Boyles, Dorota Sadowski, Scott Potter, Aleksandra Vukojicic, James Parker, William Y. Chang, Yanfei L. Ma, Mark G. Chambers, James Nelson, Barbra Barmettler, Eric M. Smith, Kara Kersjes, Evan R. Himes, Chaohua Lin, Jonathan Lucchesi, Jaladhi Brahmbhatt, Ramtin Sina, Jennifer A. Martin, Evan Maestri, Christopher M. Wiethoff, Gregory L. Dyas, Matthew D. Linnik, Songqing Na, Derrick R. Witcher, Alison Budelsky, Kira Rubtsova
×Abstract
SARS-CoV-2 mRNA vaccination generates protective B cell responses targeting the SARS-CoV-2 spike glycoprotein. Whereas anti-spike memory B cell responses are long lasting, the anti-spike humoral antibody response progressively wanes, making booster vaccinations necessary for maintaining protective immunity. Here, we qualitatively investigated the plasmablast responses by measuring from single cells within hours of sampling the affinity of their secreted antibody for the SARS-CoV-2 spike receptor binding domain (RBD) in cohorts of BNT162b2-vaccinated naive and COVID-19–recovered individuals. Using a droplet microfluidic and imaging approach, we analyzed more than 4,000 single IgG-secreting cells, revealing high interindividual variability in affinity for RBD, with variations over 4 logs. High-affinity plasmablasts were induced by BNT162b2 vaccination against Hu-1 and Omicron RBD but disappeared quickly thereafter, whereas low-affinity plasmablasts represented more than 65% of the plasmablast response at all time points. Our droplet-based method thus proves efficient at fast and qualitative immune monitoring and should be helpful for optimization of vaccination protocols.
Authors
Matteo Broketa, Aurélien Sokal, Michael Mor, Pablo Canales-Herrerias, Angga Perima, Annalisa Meola, Ignacio Fernández, Bruno Iannascoli, Guilhem Chenon, Alexis Vandenberghe, Laetitia Languille, Marc Michel, Bertrand Godeau, Sébastien Gallien, Giovanna Melica, Marija Backovic, Felix A. Rey, Jean Baudry, Natalia T. Freund, Matthieu Mahévas, Pierre Bruhns
×Abstract
The incidence of early-onset colorectal cancer (EO-CRC) is rising and is poorly understood. Lifestyle factors and altered genetic background possibly contribute. Here, we performed targeted exon sequencing of archived leukocyte DNA from 158 EO-CRC participants, which identified a missense mutation at p.A98V within the proximal DNA binding domain of Hepatic Nuclear Factor 1 α (HNF1AA98V, rs1800574). The HNF1AA98V exhibited reduced DNA binding. To test function, the HNF1A variant was introduced into the mouse genome by CRISPR/Cas9, and the mice were placed on either a high-fat diet (HFD) or high-sugar diet (HSD). Only 1% of the HNF1A mutant mice developed polyps on normal chow; however, 19% and 3% developed polyps on the HFD and HSD, respectively. RNA-Seq revealed an increase in metabolic, immune, lipid biogenesis genes, and Wnt/β-catenin signaling components in the HNF1A mutant relative to the WT mice. Mouse polyps and colon cancers from participants carrying the HNF1AA98V variant exhibited reduced CDX2 and elevated β-catenin proteins. We further demonstrated decreased occupancy of HNF1AA98V at the Cdx2 locus and reduced Cdx2 promoter activity compared with WT HNF1A. Collectively, our study shows that the HNF1AA98V variant plus a HFD promotes the formation of colonic polyps by activating β-catenin via decreasing Cdx2 expression.
Authors
Heyu Song, Ricky A. Sontz, Matthew J. Vance, Julia M. Morris, Sulaiman Sheriff, Songli Zhu, Suzann Duan, Jiping Zeng, Erika Koeppe, Ritu Pandey, Curtis A. Thorne, Elena M. Stoffel, Juanita L. Merchant
×Abstract
HIV-1 infection is characterized by inflammation and a progressive decline in CD4+ T cell count. Despite treatment with antiretroviral therapy (ART), the majority of people living with HIV (PLWH) maintain residual levels of inflammation, a low degree of immune activation, and higher sensitivity to cell death in their memory CD4+ T cell compartment. To date, the mechanisms responsible for this high sensitivity remain elusive. We have identified the transcription factor IRF-5 to be involved in impairing the maintenance of murine CD4+ T cells during chronic infection. Here, we investigate whether IRF-5 also contributes to memory CD4+ T cell loss during HIV-1 infection. We show that TLR7 and IRF-5 were upregulated in memory CD4+ T cells from PLWH, when compared with naturally protected elite controllers and HIVfree participants. TLR7 was upstream of IRF-5, promoting Caspase 8 expression in CD4+ T cells from ART HIV-1+ but not from HIVfree donors. Interestingly, the TLR7/IRF-5 axis acted synergistically with the Fas/FasL pathway, suggesting that TLR7 and IRF-5 expression in ART HIV-1+ memory CD4+ T cells represents an imprint that predisposes cells to Fas-mediated apoptosis. This predisposition could be blocked using IRF-5 inhibitory peptides, suggesting IRF-5 blockade as a possible therapy to prevent memory CD4+ T cell loss in PLWH.
Authors
Liseth Carmona-Pérez, Xavier Dagenais-Lussier, Linh T. Mai, Tanja Stögerer, Sharada Swaminathan, Stéphane Isnard, Matthew R. Rice, Betsy J. Barnes, Jean-Pierre Routy, Julien van Grevenynghe, Simona Stäger
×Abstract
Antiviral immunity often requires CD8+ cytotoxic T lymphocytes (CTLs) that actively migrate and search for virus-infected targets. Regulatory T cells (Tregs) have been shown to suppress CTL responses, but it is not known whether this is also mediated by effects on CTL motility. Here, we used intravital 2-photon microscopy in the Friend retrovirus (FV) mouse model to define the impact of Tregs on CTL motility throughout the course of acute infection. Virus-specific CTLs were very motile and had frequent short contacts with target cells at their peak cytotoxic activity. However, when Tregs were activated and expanded in late-acute FV infection, CTLs became significantly less motile and contacts with target cells were prolonged. This phenotype was associated with development of functional CTL exhaustion. Tregs had direct contacts with CTLs in vivo and, importantly, their experimental depletion restored CTL motility. Our findings identify an effect of Tregs on CTL motility as part of their mechanism of functional impairment in chronic viral infections. Future studies must address the underlying molecular mechanisms.
Authors
Daniela Mittermüller, Lucas Otto, Zoë Long, Andreas Kraus, Alexander Beer, Anja Hasenberg, Gennadiy Zelinskyy, Jaana Westmeier, Kim J. Hasenkrug, Ulf Dittmer, Matthias Gunzer
×Abstract
Patients with recurrent acute pancreatitis (RAP) are at significant risk of developing early chronic pancreatitis (CP), which progresses into irreversible, end-stage CP with severe symptoms. There is no specific therapy in RAP or in early CP that may hinder disease progression. The pathogenesis of CP is complex and involves interactions among multiple cell types, including pancreatic acinar, ductal, and stellate cells (PSC). Therefore, it is pivotal to identify common pathogenic pathways in these cells that could be targeted pharmacologically. The Orai1-mediated store-operated Ca2+ entry (SOCE) is a ubiquitous signaling mechanism that may become overactivated in pathological states resulting in intracellular Ca2+ overload. In this study, we used ex vivo and in vivo preclinical disease models to demonstrate that Orai1 inhibition prevents progression of RAP and early CP. The selective Orai1 inhibitor CM5480 restored the expression of SOCE-associated regulatory factor in acinar cells, prevented uncontrolled Ca2+ elevation, protected acinar and ductal functions, mitigated immune cell infiltration, and diminished PSC activation, proliferation, and migration. We suggest that the overactivation of Orai1 is a crucial pathogenetic event in the progression of early CP and that inhibition of Orai1 could prevent the development of end-stage CP.
Authors
Viktória Szabó, Noémi Csákány-Papp, Marietta Görög, Tamara Madácsy, Árpád Varga, Aletta Kiss, Bálint Tél, Boldizsár Jójárt, Tim Crul, Krisztina Dudás, Mária Bagyánszki, Nikolett Bódi, Ferhan Ayaydin, Shyam Jee, László Tiszlavicz, Kenneth A. Stauderman, Sudarshan Hebbar, Petra Pallagi, József Maléth
×Abstract
Human cytomegalovirus (HCMV) is the most common vertically transmitted infection worldwide, yet there are no vaccines or therapeutics to prevent congenital HCMV (cCMV) infection. Emerging evidence indicates that antibody Fc effector functions may be a previously underappreciated component of maternal immunity against HCMV. We recently reported that antibody-dependent cellular phagocytosis (ADCP) and IgG activation of FcγRI/FcγRII were associated with protection against cCMV transmission, leading us to hypothesize that additional Fc-mediated antibody functions may be important. In this same cohort of HCMV-transmitting (n = 41) and nontransmitting (n = 40) mother-infant dyads, we report that higher maternal sera antibody–dependent cellular cytotoxicity (ADCC) activation is also associated with lower risk of cCMV transmission. We investigated the relationship between ADCC and IgG responses against 9 viral antigens and found that ADCC activation correlated most strongly with sera IgG binding to the HCMV immunoevasin protein UL16. Moreover, we determined that higher UL16-specific IgG binding and FcγRIII/CD16 engagement were associated with the greatest risk reduction in cCMV transmission. Our findings indicate that ADCC-activating antibodies against targets such as UL16 may represent an important protective maternal immune response against cCMV infection that can guide future HCMV correlates studies and vaccine or antibody-based therapeutic development.
Authors
Eleanor C. Semmes, Itzayana G. Miller, Nicole Rodgers, Caroline T. Phan, Jillian H. Hurst, Kyle M. Walsh, Richard J. Stanton, Justin Pollara, Sallie R. Permar
×Abstract
Given the COVID-19 pandemic, there is interest in understanding ligand-receptor features and targeted antibody-binding attributes against emerging SARS-CoV-2 variants. Here, we developed a large-scale structure-based pipeline for analysis of protein-protein interactions regulating SARS-CoV-2 immune evasion. First, we generated computed structural models of the Spike protein of 3 SARS-CoV-2 variants (B.1.1.529, BA.2.12.1, and BA.5) bound either to a native receptor (ACE2) or to a large panel of targeted ligands (n = 282), which included neutralizing or therapeutic monoclonal antibodies. Moreover, by using the Barnes classification, we noted an overall loss of interfacial interactions (with gain of new interactions in certain cases) at the receptor-binding domain (RBD) mediated by substituted residues for neutralizing complexes in classes 1 and 2, whereas less destabilization was observed for classes 3 and 4. Finally, an experimental validation of predicted weakened therapeutic antibody binding was performed in a cell-based assay. Compared with the original Omicron variant (B.1.1.529), derivative variants featured progressive destabilization of antibody-RBD interfaces mediated by a larger set of substituted residues, thereby providing a molecular basis for immune evasion. This approach and findings provide a framework for rapidly and efficiently generating structural models for SARS-CoV-2 variants bound to ligands of mechanistic and therapeutic value.
Authors
Joseph H. Lubin, Christopher Markosian, D. Balamurugan, Minh T. Ma, Chih-Hsiung Chen, Dongfang Liu, Renata Pasqualini, Wadih Arap, Stephen K. Burley, Sagar D. Khare
×Abstract
Therapeutic strategies targeting complement have revolutionized the treatment of myasthenia gravis (MG). However, a deeper understanding of complement modulation in the human system is required to improve treatment responses and identify off-target effects shaping long-term outcomes. For this reason, we studied a cohort of patients with MG treated with either eculizumab or azathioprine as well as treatment-naive patients using a combined proteomics and metabolomics approach. This strategy validated known effects of eculizumab on the terminal complement cascade. Beyond that, eculizumab modulated the serum proteometabolome as distinct pathways were altered in eculizumab-treated patients, including the oxidative stress response, mitogen-activated protein kinase signaling, and lipid metabolism with particular emphasis on arachidonic acid signaling. We detected reduced levels of arachidonate 5-lipoxygenase (ALOX5) and leukotriene A4 in eculizumab-treated patients. Mechanistically, ligation of the C5a receptor (C5aR) is needed for ALOX5 metabolism and generation of downstream leukotrienes. As eculizumab prevents cleavage of C5 into C5a, decreased engagement of C5aR may inhibit ALOX5-mediated synthesis of pro-inflammatory leukotrienes. These findings indicate distinct off-target effects induced by eculizumab, illuminating potential mechanisms of action that may be harnessed to improve treatment outcomes.
Authors
Christopher Nelke, Christina B. Schroeter, Frauke Stascheit, Niklas Huntemann, Marc Pawlitzki, Alice Willison, Saskia Räuber, Nico Melzer, Ute Distler, Stefan Tenzer, Kai Stühler, Andreas Roos, Andreas Meisel, Sven G. Meuth, Tobias Ruck
×Abstract
While the development of different vaccines slowed the dissemination of SARS-CoV-2, the occurrence of breakthrough infections has continued to fuel the COVID-19 pandemic. To secure at least partial protection in the majority of the population through 1 dose of a COVID-19 vaccine, delayed administration of boosters has been implemented in many countries. However, waning immunity and emergence of new variants of SARS-CoV-2 suggest that such measures may induce breakthrough infections due to intermittent lapses in protection. Optimizing vaccine dosing schedules to ensure prolonged continuity in protection could thus help control the pandemic. We developed a mechanistic model of immune response to vaccines as an in silico tool for dosing schedule optimization. The model was calibrated with clinical data sets of acquired immunity to COVID-19 mRNA vaccines in healthy and immunocompromised participants and showed robust validation by accurately predicting neutralizing antibody kinetics in response to multiple doses of COVID-19 mRNA vaccines. Importantly, by estimating population vulnerability to breakthrough infections, we predicted tailored vaccination dosing schedules to minimize breakthrough infections, especially for immunocompromised individuals. We identified that the optimal vaccination schedules vary from CDC-recommended dosing, suggesting that the model is a valuable tool to optimize vaccine efficacy outcomes during future outbreaks.
Authors
Prashant Dogra, Carmine Schiavone, Zhihui Wang, Javier Ruiz-Ramírez, Sergio Caserta, Daniela I. Staquicini, Christopher Markosian, Jin Wang, H. Dirk Sostman, Renata Pasqualini, Wadih Arap, Vittorio Cristini
×Abstract
Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). Genetic mutation knockin (KI) animal models imply that altered function of the arginine-serine-rich (RS) domain is crucial for severe DCM. To test this hypothesis, we generated an RS domain deletion mouse model (Rbm20ΔRS). We showed that Rbm20ΔRS mice manifested DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 was mis-localized to the sarcoplasm in Rbm20ΔRS mouse hearts and formed RBM20 granules similar to those detected in mutation KI animals. In contrast, mice lacking the RNA recognition motif showed similar mis-splicing of major RBM20 target genes but did not develop DCM or exhibit RBM20 granule formation. Using in vitro studies with immunocytochemical staining, we demonstrated that only DCM-associated mutations in the RS domain facilitated RBM20 nucleocytoplasmic transport and promoted granule assembly. Further, we defined the core nuclear localization signal (NLS) within the RS domain of RBM20. Mutation analysis of phosphorylation sites in the RS domain suggested that this modification may be dispensable for RBM20 nucleocytoplasmic transport. Collectively, our findings revealed that disruption of RS domain–mediated nuclear localization is crucial for severe DCM caused by NLS mutations.
Authors
Yanghai Zhang, Zachery R. Gregorich, Yujuan Wang, Camila Urbano Braz, Jibin Zhang, Yang Liu, Peiheng Liu, Jiaxi Shen, Nanyumuzi Aori, Timothy A. Hacker, Henk Granzier, Wei Guo
×Abstract
T cells recognize tumor-derived mutated peptides presented on MHC by tumors. The recognition of these neo-epitopes leads to rejection of tumors, an event that is critical for successful cancer immunosurveillance. Determination of tumor-rejecting neo-epitopes in human tumors has proved difficult, though recently developed systems approaches are becoming increasingly useful at evaluating their immunogenicity. We have used the differential aggretope index to determine the neo-epitope burden of sarcomas and observed a conspicuously titrated antigenic landscape, ranging from the highly antigenic osteosarcomas to the low antigenic leiomyosarcomas and liposarcomas. We showed that the antigenic landscape of the tumors inversely reflected the historical T cell responses in the tumor-bearing patients. We predicted that highly antigenic tumors with poor antitumor T cell responses, such as osteosarcomas, would be responsive to T cell–based immunotherapy regimens and demonstrated this in a murine osteosarcoma model. Our study presents a potentially novel pipeline for determining antigenicity of human tumors, provides an accurate predictor of potential neo-epitopes, and will be an important indicator of which cancers to target with T cell–enhancing immunotherapy.
Authors
David O. Osei-Hwedieh, Abigail L. Sedlacek, Luis Mena Hernandez, Archibald Agyekum Yamoah, Swati G. Iyer, Kurt R. Weiss, Robert J. Binder
