Therapeutics
Abstract
Aneuploidy, a cancer hallmark, drives chromosomal instability, drug resistance, and clinically-aggressive tumors. Cyclin-dependent kinase 2 (CDK2) antagonism with independent inhibitors or CDK2 knock-down triggered anaphase catastrophe. This disrupts supernumerary centrosome clustering, causing multipolar division and apoptosis. Time-lapse fluorescent microscopy of FUCCI cell cycle probes transduced into aneuploid lung cancer cells revealed distinct fates of bipolar and polyploid cells after CDK2 inhibition. Apoptosis occurred in multipolar progeny but was repressed in persistent polyploid cancer cells. RNA-seq analyses after CDK2 inhibition of 4N versus 2N lung cancer cells were enriched for CDK1 pathway and KIF family members. The Cancer Genome Atlas (TCGA) analysis of lung cancers indicated CDK1 and KIF family member overexpression was associated with an unfavorable survival. Intravital microscopy of transplanted lung cancer cells in mice extended findings from the in vitro to in vivo settings. CDK2 inhibition of tumor-bearing mice produced polyploid cancer cells in vivo. These cancer cells were resistant to apoptosis and proliferated despite CDK2 inhibition. In contrast, polyploid populations were rarely detected in CDK2 inhibited human alveolar epithelial cells. These findings are translationally relevant. Combined targeting of CDK2 with CDK1 or kinesin family member antagonists should eliminate polyploid cancer cells, promote apoptosis and augment antineoplastic effects.
Authors
Liliya Tyutyunyk-Massey, Zibo Chen, Xiuxia Liu, Masanori Kawakami, Adam Harned, Yeap Ng, Brian Luke, Samuel C. Okpechi, Blessing Ogunlade, Yair Alfaro-Mora, Roberto Weigert, Kedar Narayan, Xi Liu, Ethan Dmitrovsky
Abstract
Successful allograft-specific tolerance induction would eliminate the need for daily immunosuppression and improve posttransplant quality of life. Adoptive cell therapy with regulatory T cells expressing donor-specific chimeric antigen receptors (CAR Tregs) is a promising strategy but, as monotherapy, cannot prolong survival with allografts with multiple MHC mismatches. Using an HLA-A2–transgenic haplo-mismatched heart transplantation model in immunocompetent C57BL/6 recipients, we showed that HLA-A2–specific CAR (A2.CAR) Tregs were able to synergize with a low dose of anti-CD154 to enhance graft survival. Using haplo-mismatched grafts expressing the 2W-OVA transgene and tetramer-based tracking of 2W- and OVA-specific T cells, we showed that in mice with accepted grafts, A2.CAR Tregs inhibited donor-specific T cell, B cell, and antibody responses and promoted a substantial increase in endogenous FOXP3+ Tregs with indirect donor specificity. By contrast, in mice where A2.CAR Tregs failed to prolong graft survival, FOXP3– A2.CAR T cells preferentially accumulated in rejecting allografts, and endogenous donor-specific responses were not controlled. This study therefore provides evidence for synergy between A2.CAR Tregs and CD154 blockade to promote infectious tolerance in immunocompetent recipients of haplo-mismatched heart grafts and defines features of A2.CAR Tregs when they fail to reshape host immunity toward allograft tolerance.
Authors
Samarth S. Durgam, Isaac Rosado-Sánchez, Dengping Yin, Madeleine Speck, Majid Mojibian, Ismail Sayin, Grace E. Hynes, Maria-Luisa Alegre, Megan K. Levings, Anita S. Chong
Abstract
Mitogen-activated protein kinase 8 interacting protein 3 (MAPK8IP3/JIP3) is a member of the kinesin family known to play a role in axonal transport of cargo. Mutations in the gene have been linked to severe neurodevelopmental disorders, resulting in developmental delay, intellectual disability, ataxia, tremor, autism, seizures, and visual impairment. A patient who has a missense mutation in the MAPK8IP3 gene (c. 1714 C>T, Arg578Cys) (R578C) manifests dystonia, gross motor delay and developmental delay. Here we show that the mutation is a toxic gain of function mutation which alters the interactome of JIP3, disrupts axonal transport of late endosomes, increases signaling via c-Jun N-terminal kinase (JNK), resulting in apoptosis, and disrupts the dopamine receptor 1 (D1) signaling while not affecting the dopamine receptor 2 (D2) signaling. Further, in the presence of the mutant protein, we show that 80% reduction of mutant JIP3>80% and 60% reduction of wild-type JIP3 by non-allele selective phosphorothioate (PS)-modified antisense oligonucleotides (ASOs) is well tolerated by several types of cells in vitro. Our study identifies several important new roles for JIP3 and provides important insights for therapeutic approaches, including antisense oligonucleotide reduction of JIP3.
Authors
Wei Zhang, Swapnil Mittal, Ria Thomas, Anahid Foroughishafiei, Ricardo Nunes Bastos, Wendy K. Chung, Konstantina Skourti-Stathaki, Stanley T. Crooke
Abstract
Mutations in the gap junction β2 (GJB2) gene, which encodes connexin 26, are the leading cause of genetic deafness. These mutations are characterized by the degeneration and fragmentation of gap junctions and gap junction plaques (GJPs) composed of connexin 26. Dominant-negative mutations of GJB2, such as R75W, cause syndromic hearing loss and palmoplantar keratoderma. We previously reported that the R75W mutation, a single-base substitution where C is replaced by T, causes fragmentation of GJPs. Therefore, an adenine base editor (ABE), which enables A-to-G base conversions, can potentially be useful for the treatment of this genetic disease. Here, we report that an all-in-one adeno-associated virus (AAV) vector, which includes a compact ABE (SaCas9-NNG-ABE8e) with broad targeting range, and a sgRNA targeting the R75W mutation in GJB2 corrected this pathogenic mutation and facilitated the recovery of the gap junction intercellular communication network of GJPs. In a transgenic mouse model with the GJB2 R75W mutation, AAV-mediated base editing also restored the fragmented GJPs to orderly outlines in cochlear supporting cells. Our findings suggest that an ABE-based base-editing strategy could be an optimal treatment for the dominant form of GJB2-related hearing loss, GJB2-related skin diseases, and other deafness-related mutations, especially single-base substitutions.
Authors
Takao Ukaji, Daisuke Arai, Harumi Tsutsumi, Ryoya Nakagawa, Fumihiko Matsumoto, Katsuhisa Ikeda, Osamu Nureki, Kazusaku Kamiya
Abstract
Glioblastoma (GBM) is one of the most lethal adult brain tumors with limited effective therapeutic options. Immunotherapy targeting B7-H3 (CD276) has shown promising efficacy in the treatment of gliomas. However, the response to this treatment varies among glioma patients due to individual differences. It’s necessary to find an effective strategy to improve the efficacy of targeting B7-H3 immunotherapy for non-responders. In this study, we demonstrated a strong correlation between aurora kinase A (AURKA) and CD276 expression in glioma tissue samples. Additionally, both AURKA knockdown and overexpression resulted in parallel changes in B7-H3 expression levels in glioma cells. Mechanistically, AURKA elevated B7-H3 expression by promoting epidermal growth factor receptor (EGFR) phosphorylation, which was validated in glioma cell lines and primary GBM cells. What’s more, the combination of AURKA inhibitor (alisertib) and anti-B7-H3 antibody markedly reduced tumor size and promoted CD8+ T cell infiltration and activation in mouse orthotopic syngeneic glioma models. To our knowledge, this study is the first to demonstrate AURKA-mediated B7-H3 upregulation in glioma cells; moreover, it proposes a promising therapeutic strategy combining the AURKA inhibitor alisertib with B7-H3-specific blocking mAbs.
Authors
Jinqiu Liu, Yuxuan Deng, Zhuonan Pu, Yazhou Miao, Zhaonian Hao, Herui Wang, Shaodong Zhang, Hanjie Liu, Jiejun Wang, Yifan Lv, Boyi Hu, Hong Wan, Zhengping Zhuang, Tai Sun, Shuyu Hao, Nan Ji, Jie Feng
Abstract
Tacrolimus-induced chronic nephrotoxicity (TICN) hinders its long-term use, but its mechanism remains unclear. Tacrolimus exerts its pharmacological effect by inhibiting calcineurin and its substrate NFAT. Whether the inhibition of other calcineurin substrates is related to TICN remains to be explored. Transcription factor EB (TFEB), a substrate of calcineurin, plays a crucial role in various homeostasis. Herein, we found that tacrolimus inhibited TFEB nuclear translocation and activity in mouse kidneys and HK-2 cells. Then, TFEB gain- and loss-of-function rescued the effect of tacrolimus in HK-2 cells. Furthermore, TFEB activation both by phosphorylation sites mutation and agonist rescued TICN in mice. To elucidate the mechanism of TFEB, we analyzed ChIP-seq data. Growth arrest and DNA damage-inducible 45α (GADD45α) was identified as a transcriptional target of TFEB via chromatin immunoprecipitation and dual luciferase reporter assays. And then we revealed that GADD45α overexpression rescued DNA damage and kidney injury caused by tacrolimus or TFEB knockdown in vitro, and vise versa. The protective effect of GADD45α against TICN and DNA damage was further demonstrated by overexpressing it in mice. In conclusion, the persistent inhibition of TFEB-GADD45α pathway by tacrolimus contributes to TICN. This study identifies a specific target for intervention of TICN.
Authors
Ping Gao, Xinwei Cheng, Maochang Liu, Hui Peng, Guodong Li, Tianze Shang, Jianqiao Wang, Qianyan Gao, Chenglong Zhu, Zhenpeng Qiu, Chengliang Zhang
Abstract
The proof-of-principle of the therapeutic potential of heat shock protein 47 (HSP47) for diseases characterized by defects in the collagen I synthesis is here proved in osteogenesis imperfecta (OI), a prototype of collagen disorders. Most of the OI mutations delay collagen I chains folding, increasing their exposure to post translational modifications that affect collagen secretion and impact extracellular matrix fibrils assembly. As model, we used primary fibroblasts from OI individuals with defect in the collagen prolyl-3-hydroxylation complex, since are characterized by the synthesis of homogeneously overmodified collagen molecules. We demonstrated that the exogenous recombinant HSP47 (rHSP47) is uptaken by the cells and localizes at the ER exit sites and ER Golgi intermediate compartment. rHSP47 treatment increased collagen secretion, reduced collagen post translational modifications and intracellular collagen retention and ameliorated the general ER proteostasis, leading to improved cellular homeostasis and vitality. These positive changes were also mirrored by an increased collagen content in the OI matrix. A mutation dependent effect was found in fibroblasts from three probands with collagen I mutations, for which rHSP47 was effective only in cells with the most N-term defect. A beneficial effect on bone mineralization was proved in vivo in the zebrafish p3h1-/- OI model.
Authors
Roberta Besio, Nadia Garibaldi, Alessandra Sala, Francesca Tonelli, Carla Aresi, Elisa Maffioli, Claudio Casali, Camilla Torriani, Marco Biggiogera, Simona Villani, Antonio Rossi, Gabriella Tedeschi, Antonella Forlino
Abstract
Pediatric high-grade gliomas (pHGGs) are the most aggressive brain tumors in children, necessitating innovative therapies to improve outcomes. Unlike adult gliomas, recent research reveals that childhood gliomas have distinct biological features, requiring specific treatment strategies. Here, we focused on deciphering unique genetic dependencies specific to childhood gliomas. Using a pooled CRISPR/Cas9 knockout screening approach on 65 pediatric and 10 adult high-grade glioma (HGG) cell lines, myeloid cell leukemia 1 (MCL1) emerged as a key antiapoptotic gene essential in pediatric but not adult gliomas. We demonstrated that MCL1 is targetable using current small molecule inhibitors, and its inhibition leads to potent anticancer activity across pediatric HGG cell lines irrespective of genotype. Employing predictive modeling approaches on a large set of childhood cancer cell lines with multiomics data features, we identified a potentially previously unreported cluster of CpG sites in the antiapoptotic BCL-xL/BCL2L1 gene, which predicted MCL1 inhibitor response. We extended these data across multiple pediatric tumor types, showing that BCL2L1 methylation is a broad predictor of MCL1 dependency in vitro and in vivo. Overall, our multidimensional, integrated genomic approach identified MCL1 as a promising therapeutic target in several BCL2L1-methylated pediatric cancers, offering a translational strategy to identify patients most likely to benefit from MCL1 inhibitor therapy.
Authors
Shazia Adjumain, Paul Daniel, Claire Xin Sun, Gabrielle Bradshaw, Nicole J. Chew, Vanessa Tsui, Hanbyeol Lee, Melissa Loi, Nataliya Zhukova, Dilru Habarakada, Abigail Yoel, Vijesh G. Vaghjiani, Shaye Game, Louise E. Ludlow, Naama Neeman, E. Alejandro Sweet-Cordero, David D. Eisenstat, Jason E. Cain, Ron Firestein
Abstract
Chronic wounds have emerged as a tough clinical challenge. An improved understanding of wound healing mechanisms is paramount. Collagen XVII (COL17), a pivotal constituent of hemidesmosomes, holds considerable promise for regulating epidermal cell adhesion to the basement membrane, as well as for epidermal cell motility and self-renewal of epidermal stem cells. However, the precise role of COL17 in wound repair remains elusive, and the upstream regulatory mechanisms involved have not been fully elucidated. In this study, we delineated the temporal and spatial expression patterns of COL17 at the epidermal wound edge. Subsequently, we investigated the indispensable role of COL17 in keratinocyte activation and re-epithelialization during wound healing, demonstrating the restoration of the normal repair process by COL17 overexpression in diabetic wounds. Notably, we identified a key transcriptional signaling pathway for COL17, wherein PKM2 (Pyruvate kinase isozyme M2) promotes phosphorylation of STAT3, leading to its activation and subsequent induction of COL17 expression upon injury. Ultimately, by manipulating this pathway using the PKM2 nuclear translocator SAICAR, we revealed a promising therapeutic strategy for enhancing the healing of chronic wounds.
Authors
Yangdan Liu, Chia-kang Ho, Dongsheng Wen, Jiaming Sun, Yuxin Liu, Qing-Feng Li, Yifan Zhang, Ya Gao
Abstract
The impact of remdesivir on SARS-CoV-2 diversity and evolution in vivo has remained unclear. In this single-center, retrospective cohort study, we assessed SARS-CoV-2 diversification and diversity over time in a cohort of hospitalized patients who did or did not receive remdesivir. Whole genome sequencing was performed on 98 paired specimens collected from 49 patients before and after remdesivir administration. Genetic divergence between paired specimens was not significantly different from what was observed in paired specimens from patients who did not receive the drug. However, when comparing minority variants, several positions showed preferential diversification after remdesivir treatment, several of which were associated with different variants of concern. Most notably, remdesivir administration resulted in strong selection for a nonsynonymous mutation in nsp12, G671S, previously associated with enhanced viral fitness. This same mutation was found enriched in a second cohort of 143 inpatients with specimens collected after remdesivir administration compared to controls. Only one other mutation previously implicated in remdesivir resistance (nsp12:V792I) was found to be preferentially selected for after remdesivir administration. These data suggest that SARS-CoV-2 variants with enhanced replicative fitness may be selected for in the presence of antiviral therapy as an indirect means to overcome this selective pressure.
Authors
Ted Ling-Hu, Lacy M. Simons, Estefany Rios-Guzman, Alexandre M. Carvalho, Maria Francesca R. Agnes, Arghavan Alisoltanidehkordi, Egon A. Ozer, Ramon Lorenzo-Redondo, Judd F. Hultquist
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