Therapeutics
Abstract
GM3 synthase deficiency (GM3SD) is an infantile-onset epileptic encephalopathy syndrome caused by biallelic loss-of-function mutations in ST3GAL5. Loss of ST3GAL5 activity in humans results in systemic ganglioside deficiency and severe neurological impairment. No disease-modifying treatment is currently available. Certain recombinant adeno-associated viruses (rAAVs) are capable of crossing the blood-brain barrier to induce widespread, long-term gene expression in the central nervous system (CNS), and represent a promising therapeutic strategy. Here, we show that a first-generation rAAV-ST3GAL5 replacement vector employing a ubiquitous promoter restored tissue ST3GAL5 expression and normalized cerebral gangliosides in patient-derived iPSC neurons and brain tissue from St3gal5 knock-out mice, but caused fatal hepatotoxicity when administered systemically. In contrast, a second-generation vector optimized for CNS-restricted ST3GAL5 expression, administered by either intracerebroventricular or intravenous route at postnatal day 1, allowed for safe and effective rescue of lethality and behavior impairment in symptomatic GM3SD mice up to a year. These results support further clinical development of ST3GAL5 gene therapy.
Authors
Huiya Yang, Robert Brown, Dan Wang, Kevin A. Strauss, Guangping Gao
Repurposing antipsychotic drug Amisulpride for targeting synovial fibroblast activation in arthritis
Abstract
Synovial Fibroblasts (SFs) are key pathogenic drivers in Rheumatoid arthritis (RA). Their in vivo activation by TNF is sufficient to orchestrate full arthritic pathogenesis in animal models and TNF blockade proved efficacious for a high percentage of RA patients albeit co-inducing rare but serious side effects. Aiming to find new potent therapeutics, we applied the L1000CDS2 search engine, in order to repurpose drugs that could reverse the pathogenic expression signature of arthritogenic human TNF transgenic (hTNFtg) SFs. We identified a neuroleptic drug, namely Amisulpride, which reduced SFs’ inflammatory potential while decreasing the clinical score of hTNFtg polyarthritis. Notably, we found that Amisulpride function is neither through its known targets Dopamine receptors 2 and 3 and Serotonin Receptor 7, nor through TNF-TNFRI binding inhibition. Through a click chemistry approach, novel potential targets of Amisulpride were identified, which were further validated to repress hTNFtg SFs’ inflammatory potential ex vivo (Ascc3 and Sec62), while phosphoproteomics analysis revealed that treatment altered important fibroblast activation pathways, such as adhesion. Thus, Amisulpride could prove beneficial to patients suffering from RA and the often-accompanying comorbid dysthymia, reducing SF pathogenicity along with its anti-depressive activity, serving further as a “lead” compound for the development of novel therapeutics against fibroblast activation.
Authors
Dimitra Papadopoulou, Fani Roumelioti, Christos Tzaferis, Panagiotis Chouvardas, Anna-Kathrine Pedersen, Filippos Charalampous, Eleni Christodoulou-Vafeiadou, Lydia Ntari, Niki Karagianni, Maria C. Denis, Jesper V. Olsen, Alexis Ν. Matralis, George Kollias
Abstract
Targeted biologic therapies can elicit an undesirable host immune response characterized by the development of antidrug antibodies (ADA), an important cause of treatment failure. The most widely used biologic across immune-mediated diseases is adalimumab, a tumor necrosis factor inhibitor. This study aimed to identify genetic variants that contribute to the development of ADA against adalimumab, thereby influencing treatment failure. In patients with psoriasis on their first course of adalimumab, in whom serum ADA had been evaluated 6–36 months after starting treatment, we observed a genome-wide association with ADA against adalimumab within the major histocompatibility complex (MHC). The association signal mapped to the presence of tryptophan at position 9 and lysine at position 71 of the HLA-DR peptide-binding groove, with both residues conferring protection against ADA. Underscoring their clinical relevance, these residues were also protective against treatment failure. Our findings highlight antigenic peptide presentation via MHC class II as a critical mechanism in the development of ADA against biologic therapies and downstream treatment response.
Authors
Teresa Tsakok, Jake Saklatvala, Theo Rispens, Floris C. Loeff, Annick de Vries, Michael H. Allen, Ines A. Barbosa, David Baudry, Tejus Dasandi, Michael Duckworth, Freya Meynell, Alice Russell, Anna Chapman, Sandy McBride, Kevin McKenna, Gayathri Perera, Helen Ramsay, Raakhee Ramesh, Kathleen Sands, Alexa Shipman, the Biomarkers of Systemic Treatment Outcomes in Psoriasis (BSTOP) Study Group, A. David Burden, Christopher E.M. Griffiths, Nick J. Reynolds, Richard B. Warren, Satveer Mahil, Jonathan Barker, Nick Dand, Catherine Smith, Michael A. Simpson
Abstract
Diabetes is associated with increased risk for kidney and liver diseases, congestive heart failure, and mortality. Urinary glucose excretion using sodium-glucose cotransporter 2 (SGLT2) inhibitors prevents these adverse outcomes. We performed in vivo metabolic labeling with 13C-glucose in normoglycemic and diabetic mice treated with or without the SGLT2 inhibitor dapagliflozin, followed by simultaneous metabolomics and metabolic flux analyses in different organs and the plasma. We found that in diabetes, glycolysis and glucose oxidation are impaired in the kidney, liver, and heart. Treatment with dapagliflozin failed to rescue glycolysis and further inhibited pyruvate kinase activity in the liver. SGLT2 inhibition increased glucose oxidation in all organs; in the kidney, this effect was associated with modulation of the redox state, which may protect against oxidative stress. In addition, diabetes was associated with altered methionine cycle metabolism, evident by decreased betaine and methionine levels, whereas treatment with SGLT2i increased hepatic betaine along with decreased homocysteine levels. mTORC1 activity was inhibited by SGLT2i along with stimulation of AMPK in both normoglycemic and diabetic animals, possibly explaining the protective effects against kidney, liver, and heart diseases. Collectively, our findings suggest that SGLT2i induces metabolic reprogramming orchestrated by AMPK-mTORC1 signaling with common and distinct effects in various tissues with implications for diabetes and aging.
Authors
Aviram Kogot-Levin, Yael Riahi, Ifat Abramovich, Ofri Mosenzon, Bella Agranovich, Liat Kadosh, Rachel Ben-Haroush Schyr, Doron Kleiman, Liad Hinden, Erol Cerasi, Danny Ben-Zvi, Ernesto Bernal-Mizrachi, Joseph Tam, Eyal Gottlieb, Gil Leibowitz
Abstract
TGF-β signaling is crucial for modulating osteoarthritis (OA), and protein phosphatase magnesium–dependent 1A (PPM1A) has been reported as a phosphatase of SMAD2 and regulates TGF-β signaling, while the role of PPM1A in cartilage homeostasis and OA development remains largely unexplored. In this study, we found increased PPM1A expression in OA chondrocytes and confirmed the interaction between PPM1A and phospho-SMAD2 (p-SMAD2). Importantly, our data show that PPM1A KO substantially protected mice treated with destabilization of medial meniscus (DMM) surgery against cartilage degeneration and subchondral sclerosis. Additionally, PPM1A ablation reduced the cartilage catabolism and cell apoptosis after the DMM operation. Moreover, p-SMAD2 expression in chondrocytes from KO mice was higher than that in WT controls with DMM induction. However, intraarticular injection with SD-208, repressing TGF-β/SMAD2 signaling, dramatically abolished protective phenotypes in PPM1A-KO mice. Finally, a specific pharmacologic PPM1A inhibitor, Sanguinarine chloride (SC) or BC-21, was able to ameliorate OA severity in C57BL/6J mice. In summary, our study identified PPM1A as a pivotal regulator of cartilage homeostasis and demonstrated that PPM1A inhibition attenuates OA progression via regulating TGF-β/SMAD2 signaling in chondrocytes and provided PPM1A as a potential target for OA treatment.
Authors
Qinwen Ge, Zhenyu Shi, Kai-ao Zou, Jun Ying, Jiali Chen, Wenhua Yuan, Weidong Wang, Luwei Xiao, Xia Lin, Di Chen, Xin-Hua Feng, Ping-er Wang, Peijian Tong, Hongting Jin
Abstract
Despite advances in ovarian cancer (OC) therapy, recurrent OC remains a poor-prognosis disease. Because of the close interaction between OC cells and the tumor microenvironment (TME), it is important to develop strategies that target tumor cells and engage components of the TME. A major obstacle in the development of OC therapies is the identification of targets with expression limited to tumor surface to avoid off-target interactions. The follicle-stimulating hormone receptor (FSHR) has selective expression on ovarian granulosa cells and is expressed on 50%–70% of serous OCs. We generated mAbs targeting the external domain of FSHR using in vivo–expressed FSHR vector. By high-throughput flow analysis, we identified multiple clones and downselected D2AP11, a potent FSHR surface–targeted mAb. D2AP11 identifies important OC cell lines derived from tumors with different mutations, including BRCA1/2, and lines resistant to a wide range of therapies. We used D2AP11 to develop a bispecific T cell engager. In vitro addition of PBMCs and T cells to D2AP11-TCE induced specific and potent killing of different genetic and immune escape OC lines, with EC50s in the ng/ml range, and attenuated tumor burden in OC-challenged mouse models. These studies demonstrate the potential utility of biologics targeting FSHR for OC and perhaps other FSHR-positive cancers.
Authors
Devivasha Bordoloi, Pratik S. Bhojnagarwala, Alfredo Perales-Puchalt, Abhijeet J. Kulkarni, Xizhou Zhu, Kevin Liaw, Ryan P. O’Connell, Daniel H. Park, Daniel W. Kulp, Rugang Zhang, David B. Weiner
Abstract
The aberrant activation of STAT3 is associated with the etiology and progression in a variety of malignant epithelial-derived tumors, including head and neck squamous cell carcinoma (HNSCC) and colorectal cancer (CRC). Due to the lack of an enzymatic catalytic site or a ligand-binding pocket, there are no small-molecule inhibitors directly targeting STAT3 that have been approved for clinical translation. Emerging proteolysis targeting chimeric (PROTAC) technology–based approach represents a potential strategy to overcome the limitations of conventional inhibitors and inhibit activation of STAT3 and downstream genes. In this study, the heterobifunctional small-molecule–based PROTACs are successfully prepared from toosendanin (TSN), with 1 portion binding to STAT3 and the other portion binding to an E3 ubiquitin ligase. The optimized lead PROTAC (TSM-1) exhibits superior selectivity, potency, and robust antitumor effects in STAT3-dependent HNSCC and CRC — especially in clinically relevant patient-derived xenografts (PDX) and patient-derived organoids (PDO). The following mechanistic investigation identifies the reduced expression of critical downstream STAT3 effectors, through which TSM-1 promotes cell cycle arrest and apoptosis in tumor cells. These findings provide the first demonstration to our knowledge of a successful PROTAC-targeting strategy in STAT3-dependent epithelial cancer.
Authors
Jinmei Jin, Yaping Wu, Zeng Zhao, Ye Wu, Yu-dong Zhou, Sanhong Liu, Qingyan Sun, Guizhu Yang, Jiayi Lin, Dale G. Nagle, Jiangjiang Qin, Zhiyuan Zhang, Hong-zhuan Chen, Weidong Zhang, Shuyang Sun, Xin Luan
Abstract
HIV-specific chimeric antigen receptor–T cell (CAR T cell) therapies are candidates to functionally cure HIV infection in people with HIV (PWH) by eliminating reactivated HIV-infected cells derived from latently infected cells within the HIV reservoir. Paramount to translating such therapeutic candidates successfully into the clinic will require anti-HIV CAR T cells to localize to lymphoid tissues in the body and eliminate reactivated HIV-infected cells such as CD4+ T cells and monocytes/macrophages. Here we show that i.v. injected anti-HIV duoCAR T cells, generated using a clinical-grade anti-HIV duoCAR lentiviral vector, localized to the site of active HIV infection in the spleen of humanized mice and eliminated HIV-infected PBMCs. CyTOF analysis of preinfusion duoCAR T cells revealed an early memory phenotype composed predominantly of CCR7+ stem cell–like/central memory T cells (TSCM/TCM) with expression of some effector-like molecules. In addition, we show that anti-HIV duoCAR T cells effectively sense and kill HIV-infected CD4+ T cells and monocytes/macrophages. Furthermore, we demonstrate efficient genetic modification of T cells from PWH on suppressive ART into anti-HIV duoCAR T cells that subsequently kill autologous PBMCs superinfected with HIV. These studies support the safety and efficacy of anti-HIV duoCAR T cell therapy in our presently open phase I/IIa clinical trial (NCT04648046).
Authors
Kim Anthony-Gonda, Alex Ray, Hang Su, Yuge Wang, Ying Xiong, Danica Lee, Ariele Block, Vanessa Chilunda, Jessica Weiselberg, Lily Zemelko, Yen Y. Wang, Sarah Kleinsorge-Block, Jane S. Reese, Marcos de Lima, Christina Ochsenbauer, John C. Kappes, Dimiter S. Dimitrov, Rimas Orentas, Steven G. Deeks, Rachel L. Rutishauser, Joan W. Berman, Harris Goldstein, Boro Dropulić
Abstract
The folding and trafficking of transmembrane glycoproteins are essential for cellular homeostasis and compromised in many diseases. In Niemann-Pick type C disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, the transmembrane glycoprotein NPC1 misfolds due to disease-causing missense mutations. While mutant NPC1 has emerged as a robust target for proteostasis modulators, these drug development efforts have been unsuccessful in mouse models. Here, we demonstrate unexpected differences in trafficking through the medial Golgi between mouse and human I1061T-NPC1, a common disease-causing mutant. We establish that these distinctions are governed by differences in the NPC1 protein sequence rather than by variations in the ER folding environment. Moreover, we demonstrate direct effects of mutant protein trafficking on the response to small molecules that modulate the endoplasmic reticulum folding environment by affecting Ca++ concentration. Finally, we develop a panel of isogenic human NPC1 iNeurons expressing wild type, I1061T-, and R934L-NPC1 and demonstrate their utility in testing these candidate therapeutics. Our findings identify important rules governing mutant NPC1’s response to proteostatic modulators and highlight the importance of species- and mutation-specific responses for therapy development.
Authors
Mark L. Schultz, Kylie J. Schache, Ruth D. Azaria, Esmée Q. Kuiper, Steven Erwood, Evgueni A. Ivakine, Nicole Y. Farhat, Forbes D. Porter, Koralege C. Pathmasiri, Stephanie M. Cologna, Michael D. Uhler, Andrew P. Lieberman
Abstract
Pancreatic neuroendocrine tumors (PNETs) are malignancies arising from the islets of Langerhans. Therapeutic options are limited for the over 50% of patients who present with metastatic disease. We aimed to identify mechanisms to remodel the PNET tumor microenvironment (TME) to ultimately enhance susceptibility to immunotherapy. The TMEs of localized and metastatic PNETs were investigated using an approach that combines RNA-sequencing, cancer and T cell profiling, and pharmacologic perturbations. RNA-sequencing analysis indicated that the primary tumors of metastatic PNETs showed significant activation of inflammatory and immune-related pathways. We determined that metastatic PNETs featured increased numbers of tumor-infiltrating T cells compared to localized tumors. T cells isolated from both localized and metastatic PNETs showed evidence of recruitment and antigen-dependent activation, suggestive of an immune-permissive microenvironment. A computational analysis suggested that vorinostat, a histone deacetylase inhibitor, may perturb the transcriptomic signature of metastatic PNETs. Treatment of PNET cell lines with vorinostat increased chemokine CCR5 expression by NF-κB activation. Vorinostat treatment of patient-derived metastatic PNET tissues augmented recruitment of autologous T cells, which was substantiated in a mouse model of PNET. Pharmacologic induction of chemokine expression may represent a promising approach for enhancing the immunogenicity of metastatic PNET TMEs.
Authors
Jacques Greenberg, Jessica Limberg, Akanksha Verma, David Kim, Xiang Chen, Yeon J. Lee, Maureen D. Moore, Timothy M. Ullmann, Jessica W. Thiesmeyer, Zachary Loewenstein, Kevin J. Chen, Caitlin E. Egan, Dessislava Stefanova, Rohan Bareja, Rasa Zarnegar, Brendan M. Finnerty, Theresa Scognamiglio, Yi-Chieh Nancy Du, Olivier Elemento, Thomas J. Fahey III, Irene M. Min
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