Nephrolithiasis is a common and recurrent disease affecting 9% of the US population. Hyperoxaluria is major risk factor for calcium oxalate kidney stones, which constitutes two-thirds of all kidney stones. Gastrointestinal epithelia play an important role in oxalate handling due to the presence of SLC26A family anion exchangers that facilitate oxalate transport. SLC26A3 (originally named DRA, down-regulated in adenoma) is an anion exchanger of chloride, bicarbonate and oxalate thought to facilitate intestinal oxalate absorption, as evidenced by ~70% reduced urine oxalate excretion in knock-out mice. We previously identified, by high-throughput screening and medicinal chemistry, a small molecule SLC26A3 inhibitor (DRAinh-A270) that selectively inhibited SLC26A3-mediated chloride/bicarbonate exchange (IC50 ~ 35 nM), and, as found here, oxalate/chloride exchange (IC50 ~ 60 nM). In colonic closed loops in mice, luminal DRAinh-A270 inhibited oxalate absorption by 70%. In a model of acute hyperoxaluria produced by oral sodium oxalate loading, DRAinh-A270 largely prevented the ~2.5-fold increase in urine oxalate/creatinine ratio. In a mouse model of oxalate nephropathy produced by a high-oxalate low-calcium diet, vehicle-treated mice by day 14 developed marked hyperoxaluria with elevated serum creatinine, renal calcium oxalate crystal deposition and renal injury, which were largely prevented by DRAinh-A270 (10 mg/kg twice daily). DRAinh-A270 administered over 7 days to healthy mice did not show significant toxicity as assessed by CBC, serum chemistries and tissue histology. Our findings support a major role of SLC26A3 in intestinal oxalate absorption and suggest the therapeutic utility of SLC26A3 inhibition for treatment of hyperoxaluria and prevention of calcium oxalate nephrolithiasis.
Onur Cil, Qi Tifany Chu, Sujin Lee, Peter M. Haggie, Alan S. Verkman
Cytokine therapy is limited by undesirable off-target side effects as well as terminal differentiation and exhaustion of chronically stimulated T cells. Here, we describe the signaling properties of a potentially unique cytokine by design, where T cell surface binding and signaling are separated between 2 different families of receptors. This fusion protein cytokine, called OMCPmutIL-2, bound with high affinity to the cytotoxic lymphocyte-defining immunoreceptor NKG2D but signaled through the common γ chain cytokine receptor. In addition to precise activation of cytotoxic T cells due to redirected binding, OMCPmutIL-2 resulted in superior activation of both human and murine CD8+ T cells by improving their survival and memory cell generation and decreasing exhaustion. This functional improvement was the direct result of altered signal transduction based on the reorganization of surface membrane lipid rafts that led to Janus kinase-3–mediated phosphorylation of the T cell receptor rather than STAT/AKT signaling intermediates. This potentially novel signaling pathway increased CD8+ T cell response to low-affinity antigens, activated nuclear factor of activated T cells transcription factors, and promoted mitochondrial biogenesis. OMCPmutIL-2 thus outperformed other common γ chain cytokines as a catalyst for in vitro CD8+ T cell expansion and in vivo CD8+ T cell–based immunotherapy.
Anirban Banerjee, Dongge Li, Yizhan Guo, Zhongcheng Mei, Christine Lau, Kelly Chen, John Westwick, Jeffery B. Klauda, Adam Schrum, Eric R. Lazear, Alexander S. Krupnick
Cyclophosphamide (CPA) and doxorubicin (DOX) are key components of chemotherapy for triple-negative breast cancer (TNBC) although suboptimal outcomes are commonly associated with drug resistance and/or intolerable side-effects. Through an approach combining high-throughput screening and chemical modification, we developed CN06 as a dual activator of the constitutive androstane receptor (CAR) and nuclear factor erythroid 2-related factor 2 (Nrf2). CN06 enhances CAR-induced bioactivation of CPA (a prodrug) by provoking hepatic expression of CYP2B6, while repressing DOX-induced cytotoxicity in cardiomyocytes in vitro via stimulating Nrf2-antioxidant signaling. Utilizing a multicellular co-culture model incorporating human primary hepatocytes, TNBC cells, and cardiomyocytes, we show that CN06 increased CPA/DOX-mediated TNBC cell death via CAR-dependent CYP2B6 induction and subsequent conversion of CPA to its active metabolite 4-hydroxy-CPA, while protecting against DOX-induced cardiotoxicity by selectively activating Nrf2-antioxidant signaling in cardiomyocytes but not in TNBC cells. Further, CN06 preserves the viability and function of human iPSC-derived cardiomyocytes by modulating antioxidant defenses, decreasing apoptosis, and enhancing the kinetics of contraction and relaxation. Collectively, our findings identify CAR and Nrf2 as novel combined therapeutic targets whereby CN06 holds the potential to improve the efficacy:toxicity ratio of CPA/DOX-containing chemotherapy.
Sydney Stern, Dongdong Liang, Linhao Li, Ritika Kurian, Caitlin Lynch, Srilatha Sakamuru, Scott Heyward, Junran Zhang, Kafayat Ajoke Kareem, Young Wook Chun, Ruili Huang, Menghang Xia, Charles C. Hong, Fengtian Xue, Hongbing Wang
In situ vaccination has demonstrated the feasibility of priming local immunity for systemic antitumor responses. Although direct intratumoral delivery of adjuvant is the mainstay, tumor-draining lymph nodes (TDLNs) also play essential roles in antitumor immunity. We report that directing an adjuvant to both tumors and TDLNs during in situ vaccination can induce robust antitumor responses. Conventional intratumoral dosing leads to tumor-limited delivery of agents; however, delivery to both tumors and TDLNs can be ensured through a micellar formation. The peritumoral delivery of micellar MEDI9197 (mcMEDI), a toll-like receptor 7/8 agonist, induced significantly stronger innate and adaptive immune responses than those on conventional dosing. Optimal dosing was crucial because excessive or insufficient accumulation of the adjuvant in the TDLNs compromised therapeutic efficacy. The combination of local mcMEDI therapy significantly improved the efficacy of systemic anti-programmed death receptor-1 therapy. These data suggest that rerouting adjuvants to tumors and TDLNs can augment the therapeutic efficacy of in situ vaccination.
Moonkyoung Jeong, Heegon Kim, Junyong Yoon, Dong-Hyun Kim, Ji-Ho Park
Understanding the reorganization of neural circuits spared after spinal cord injury in the motor cortex and spinal cord would provide insight for developing therapeutics. Using optogenetic mapping we demonstrate a transhemispheric recruitment of neural circuits in the contralateral cortical M1/M2 area to improve the impaired forelimb function after a cervical 5 right-sided hemisection in mice, a model mimicking the human Brown-Séquard syndrome. This cortical reorganization can be elicited by a selective cortical optogenetic neuromodulation paradigm. Areas of whisker, jaw, and neck, together with the rostral forelimb area, on the motor cortex ipsilateral to the lesion are engaged to control the ipsilesional forelimb in both stimulation and non-stimulation groups at 8 weeks post-injury. However, significant functional benefits are only seen in the stimulation group. Using anterograde tracer, we further reveal a robust sprouting of the intact corticospinal tract in the spinal cord of those animals receiving optogenetic stimulation. The intraspinal cortical spinal axonal sprouting corelates with the forelimb functional recovery. Thus, specific neuromodulation of the cortical neural circuits induces massive neural reorganization both in the motor cortex and spinal cord, constructing an alternative motor pathway in restoring impaired forelimb function.
Wei Wu, Tyler Nguyen, Josue D. Ordaz, Yi Ping Zhang, Nai-Kui Liu, Xinhua Hu, Yuxiang Liu, Xingjie Ping, Qi Han, Xiangbing Wu, Wenrui Qu, Sujuan Gao, Christopher B. Shields, Xiaoming Jin, Xiao-Ming Xu
Most therapeutic monoclonal antibodies target the receptor-binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Unfortunately, the RBD is a hot spot for mutations in SARS-CoV-2 variants, which will lead to loss of the neutralizing function of current therapeutic monoclonal antibodies. Universal monoclonal antibodies for different variants are necessary. We identified monoclonal antibodies that recognized the S2 region of the spike protein, which is identical in different variants. The monoclonal antibodies could neutralize SARS-CoV-2 infection and protect animals from SARS-CoV-2 challenge. After cloning the variable region of the light chain and heavy chain, the variable region sequences were humanized to select a high-affinity humanized monoclonal antibody (hMab5.17). hMab5.17 protected animals from SARS-CoV-2 challenge and neutralized SARS-CoV-2 variant infection. We further identified the linear epitope of the monoclonal antibody, which is not mutated in any variant of concern (VOC). These data suggest that a monoclonal antibody recognizing the S2 region of the spike protein will be a potential universal therapeutic monoclonal antibody for COVID-19.
Wan-Ling Wu, Chen-Yi Chiang, Szu-Chia Lai, Chia-Yi Yu, Yu-Ling Huang, Hung-Chun Liao, Ching-Len Liao, Hsin-Wei Chen, Shih-Jen Liu
Standard-of-care treatment for advanced HER2+ breast cancers (BC) is comprised of two HER2-specific monoclonal antibodies (mAb), Trastuzumab (T) and Pertuzumab (P) with chemotherapy. While this combination (T+P) is highly effective, its synergistic mechanism of action (MOA) is not completely known. Initial studies had demonstrated that Pertuzumab suppressed HER2 hetero-dimerization as the potential therapeutic MOA, thus the improved outcome associated with the T+P combination MOA compared to Trastuzumab alone has been widely reported as being due to Pertuzumab-mediated suppression of HER2 signaling in combination with Trastuzumab-mediated induction of anti-tumor immunity. Unraveling this MOA may be critical to extend this combination strategy to other antigens or other cancers, as well as improving this current treatment modality. Using novel murine and human versions of Pertuzumab, we found it induced both Antibody-Dependent-Cellular-Phagocytosis (ADCP) by tumor-associated macrophages and suppression of HER2 oncogenic signaling. Most significantly, we identified that only T+P combination therapy, but not when either antibody used in isolation, allows for the activation of the classical complement pathway, resulting in both direct complement-dependent cytotoxicity (CDC) as well as complement-dependent cellular phagocytosis (CDCP) of HER2+ BC cells. Notably, we show that tumor expression of C1q was positively associated with survival outcome in HER2+ BC patients, whereas expression of complement regulators CD55 and CD59 were inversely correlated, suggesting the importance of complement activity in clinical outcomes. Accordingly, inhibition of C1 activity in mice abolished the synergistic therapeutic activity of T+P therapy, whereas knockdown of CD55 and CD59 expression enhanced T+P efficacy. In summary, our study identifies classical complement activation as a significant anti-tumor MOA for T+P therapy that may be functionally enhanced to augment therapeutic efficacy in the clinic.
Li-Chung Tsao, Erika J. Crosby, Timothy N. Trotter, Junping Wei, Tao Wang, Xiao Yang, Amanda N. Summers, Gangjun Lei, Christopher A. Rabiola, Lewis A. Chodosh, William J. Muller, Herbert Kim Lyerly, Zachary C. Hartman
Mucosal healing is a key treatment goal for inflammatory bowel disease, and adequate epithelial regeneration is required for an intact gut epithelium. However, the underlying mechanism is unclear. Long non-coding RNAs (lncRNAs) have been reported to be involved in the development of inflammatory bowel disease. Here, we report that a lncRNA named Gm31629, decreases in intestinal epithelial cells in response to inflammatory stimulation. Gm31629 deficiency leads to exacerbated intestinal inflammation and delayed epithelial regeneration in dextran sulfate sodium (DSS) -induced colitis model. Mechanistically, Gm31629 promotes E2F pathways and cell proliferation by stabilizing Y-box protein 1 (YB-1), thus facilitating epithelial regeneration. Genetic overexpression of Gm31629 protects against DSS-induced colitis in vivo. Theaflavin 3-gallate, a natural compound mimicking Gm31629, alleviates DSS-induced epithelial inflammation and mucosal damage. These results demonstrate an essential role of lncRNA Gm31629 in linking intestinal inflammation and epithelial cell proliferation, providing a potential therapeutic approach to inflammatory bowel disease.
Xu Feng, Ye Xiao, Jian He, Mi Yang, Qi Guo, Tian Su, Yan Huang, Jun Yi, Chang-Jun Li, Xiang-Hang Luo, Xiao-Wei Liu, Hai-Yan Zhou
Prion protein (PrP) concentration controls the kinetics of prion replication and is a genetically and pharmacologically validated therapeutic target for prion disease. In order to evaluate PrP concentration as a pharmacodynamic biomarker and assess its contribution to known prion disease risk factors, we developed and validated a plate-based immunoassay reactive for PrP across six species of interest and applicable to brain and cerebrospinal fluid (CSF). PrP concentration varies dramatically between different brain regions in mice, cynomolgus macaques, and humans. PrP expression does not appear to contribute to the known risk factors of age, sex, or common PRNP genetic variants. CSF PrP is lowered in the presence of rare pathogenic PRNP variants, with heterozygous carriers of P102L displaying 55% and of D178N just 31% the CSF PrP concentration of mutation-negative controls. In rodents, pharmacologic reduction of brain Prnp RNA is reflected in brain parenchyma PrP, and in turn in CSF PrP, validating CSF as a sampling compartment for the effect of PrP-lowering therapy. Our findings support the use of CSF PrP as a pharmacodynamic biomarker for PrP-lowering drugs, and suggest that relative reduction from individual baseline CSF PrP concentration may be an appropriate marker for target engagement.
Meredith A. Mortberg, Hien T. Zhao, Andrew G. Reidenbach, Juliana E. Gentile, Eric Kuhn, Jill O'Moore, Patrick M. Dooley, Theresa R. Connors, Curt Mazur, Shona W. Allen, Bianca A. Trombetta, Alison J. McManus, Matthew R. Moore, Jiewu Liu, Deborah E. Cabin, Holly B. Kordasiewicz, Joel Mathews, Steven E. Arnold, Sonia M. Vallabh, Eric Vallabh Minikel
The G-protein coupled C-X-C chemokine receptor 4 (CXCR4) is a candidate therapeutic target for tissue fibrosis. A novel fully human single-domain antibody-like scaffold i-body AD-114-PA600 (AD-114) with specific high binding affinity to CXCR4 has been developed. To define the renoprotective role, AD-114 was administrated in a mouse model of renal fibrosis induced by folic acid (FA). Increased extracellular matrix (ECM) accumulation, macrophage infiltration, inflammatory response, transforming growth factor beta-1 (TGF-β1) expression and fibroblasts activation were observed in kidneys of mice with FA-induced nephropathy. These markers were normalized or partially reversed by AD-114 treatment. In vitro studies demonstrated AD-114 blocked TGF-β1-induced upregulated expression of ECM, matrix metallopeptidase-2 (MMP-2) and downstream p38 mitogen-activated protein kinases (p38 MAPK) and Phosphoinositide 3-kinases (PI3K)/AKT/ mammalian target of rapamycin (mTOR) signaling pathways in a renal proximal tubular cell line. Additionally, these renoprotective effects were validated in a second model of unilateral ureteral obstruction (UUO) using a second generation of AD-114 (Fc-fused AD-114, also named AD-214). Collectively, these results suggest a renoprotective role of AD-114 as it inhibited the chemotactic function of CXCR4 as well as blocked CXCR4 downstream p38 MAPK and PI3K/AKT/mTOR signaling, which establish a therapeutic strategy for AD-114 targeting CXCR4 to limit renal fibrosis.
Qinghua Cao, Chunling Huang, Hao Yi, Anthony J. Gill, Angela Chou, Michael Foley, Chris G. Hosking, Kevin K. Lim, Cristina F. Triffon, Ying Shi, Xin-Ming Chen, Carol A. Pollock
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