Although tyrosine kinase inhibitor (TKI) therapy shows marked clinical efficacy in patients with anaplastic lymphoma kinase–positive (ALK+) and ROS proto-oncogene 1–positive (ROS1+) non–small cell lung cancer (NSCLC), most of these patients eventually relapse with acquired resistance. Therefore, genome-wide CRISPR/Cas9 knockout screening was performed using an ALK+ NSCLC cell line established from pleural effusion without ALK-TKI treatment. After 9 days of ALK-TKI therapy, sequencing analysis was performed, which identified several tumor suppressor genes, such as NF2 or MED12, and multiple candidate genes. Among them, this study focused on ERRFI1, which is known as MIG6 and negatively regulates EGFR signaling. Interestingly, MIG6 loss induced resistance to ALK-TKIs by treatment with quite a low dose of EGF, which is equivalent to plasma concentration, through the upregulation of MAPK and PI3K/AKT/mTOR pathways. Combination therapy with ALK-TKIs and anti-EGFR antibodies could overcome the acquired resistance in both in vivo and in vitro models. In addition, this verified that MIG6 loss induces resistance to ROS1-TKIs in ROS1+ cell lines. This study found a potentially novel factor that plays a role in ALK and ROS1-TKI resistance by activating the EGFR pathway with low-dose ligands.
Nobuyuki Kondo, Takahiro Utsumi, Yuki Shimizu, Ai Takemoto, Tomoko Oh-hara, Ken Uchibori, Sophia Subat-Motoshi, Hironori Ninomiya, Kengo Takeuchi, Makoto Nishio, Yasunari Miyazaki, Ryohei Katayama
Previous studies have implicated the orexigenic hormone ghrelin as a mediator of exercise endurance and the feeding response postexercise. Specifically, plasma ghrelin levels nearly double in mice when they are subjected to an hour-long bout of high-intensity interval exercise (HIIE) using treadmills. Also, growth hormone secretagogue receptor–null (GHSR-null) mice exhibit decreased food intake following HIIE and diminished running distance (time until exhaustion) during a longer, stepwise exercise endurance protocol. To investigate whether ghrelin-responsive mediobasal hypothalamus (MBH) neurons mediate these effects, we stereotaxically delivered the inhibitory designer receptor exclusively activated by designer drugs virus AAV2-hSyn-DIO-hM4(Gi)-mCherry to the MBH of Ghsr-IRES-Cre mice, which express Cre recombinase directed by the Ghsr promoter. We found that chemogenetic inhibition of GHSR-expressing MBH neurons (upon delivery of clozapine-N-oxide) 1) suppressed food intake following HIIE, 2) reduced maximum running distance and raised blood glucose and blood lactate levels during an exercise endurance protocol, 3) reduced food intake following ghrelin administration, and 4) did not affect glucose tolerance. Further, HIIE increased MBH Ghsr expression. These results indicate that activation of ghrelin-responsive MBH neurons is required for the normal feeding response to HIIE and the usual amount of running exhibited during an exercise endurance protocol.
Omprakash Singh, Sean B. Ogden, Salil Varshney, Kripa Shankar, Deepali Gupta, Subhojit Paul, Sherri Osborne-Lawrence, Corine P. Richard, Nathan P. Metzger, Connor Lawrence, Luis Leon Mercado, Jeffrey M. Zigman
Transmembrane and tetratricopeptide repeat 4 (Tmtc4) is a deafness gene in mice. Tmtc4-KO mice have rapidly progressive postnatal hearing loss due to overactivation of the unfolded protein response (UPR); however, the cellular basis and human relevance of Tmtc4-associated hearing loss in the cochlea was not heretofore appreciated. We created a hair cell–specific conditional KO mouse that phenocopies the constitutive KO with postnatal onset deafness, demonstrating that Tmtc4 is a hair cell–specific deafness gene. Furthermore, we identified a human family in which Tmtc4 variants segregate with adult-onset progressive hearing loss. Lymphoblastoid cells derived from multiple affected and unaffected family members, as well as human embryonic kidney cells engineered to harbor each of the variants, demonstrated that the human Tmtc4 variants confer hypersensitivity of the UPR toward apoptosis. These findings provide evidence that TMTC4 is a deafness gene in humans and further implicate the UPR in progressive hearing loss.
Jiang Li, Byung Yoon Choi, Yasmin Eltawil, Noura Ismail Mohamad, Yesai Park, Ian R. Matthews, Jin Hee Han, Bong Jik Kim, Elliott H. Sherr, Dylan K. Chan
Fibroblast growth factor 23 (FGF23) is a phosphate-regulating (Pi-regulating) hormone produced by bone. Hereditary hypophosphatemic disorders are associated with FGF23 excess, impaired skeletal growth, and osteomalacia. Blocking FGF23 became an effective therapeutic strategy in X-linked hypophosphatemia, but testing remains limited in autosomal recessive hypophosphatemic rickets (ARHR). This study investigates the effects of Pi repletion and bone-specific deletion of Fgf23 on bone and mineral metabolism in the dentin matrix protein 1–knockout (Dmp1KO) mouse model of ARHR. At 12 weeks, Dmp1KO mice showed increased serum FGF23 and parathyroid hormone levels, hypophosphatemia, impaired growth, rickets, and osteomalacia. Six weeks of dietary Pi supplementation exacerbated FGF23 production, hyperparathyroidism, renal Pi excretion, and osteomalacia. In contrast, osteocyte-specific deletion of Fgf23 resulted in a partial correction of FGF23 excess, which was sufficient to fully restore serum Pi levels but only partially corrected the bone phenotype. In vitro, we show that FGF23 directly impaired osteoprogenitors’ differentiation and that DMP1 deficiency contributed to impaired mineralization independent of FGF23 or Pi levels. In conclusion, FGF23-induced hypophosphatemia is only partially responsible for the bone defects observed in Dmp1KO mice. Our data suggest that combined DMP1 repletion and FGF23 blockade could effectively correct ARHR-associated mineral and bone disorders.
Guillaume Courbon, Dominik Kentrup, Jane Joy Thomas, Xueyan Wang, Hao-Hsuan Tsai, Jadeah Spindler, John Von Drasek, Laura Mazudie Ndjonko, Marta Martinez-Calle, Sana Lynch, Lauriane Hivert, Xiaofang Wang, Wenhan Chang, Jian Q. Feng, Valentin David, Aline Martin
Monocyte-derived macrophages, the major source of pathogenic macrophages in COVID-19, are oppositely instructed by macrophage CSF (M-CSF) or granulocyte macrophage CSF (GM-CSF), which promote the generation of antiinflammatory/immunosuppressive MAFB+ (M-MØ) or proinflammatory macrophages (GM-MØ), respectively. The transcriptional profile of prevailing macrophage subsets in severe COVID-19 led us to hypothesize that MAFB shapes the transcriptome of pulmonary macrophages driving severe COVID-19 pathogenesis. We have now assessed the role of MAFB in the response of monocyte-derived macrophages to SARS-CoV-2 through genetic and pharmacological approaches, and we demonstrate that MAFB regulated the expression of the genes that define pulmonary pathogenic macrophages in severe COVID-19. Indeed, SARS-CoV-2 potentiated the expression of MAFB and MAFB-regulated genes in M-MØ and GM-MØ, where MAFB upregulated the expression of profibrotic and neutrophil-attracting factors. Thus, MAFB determines the transcriptome and functions of the monocyte-derived macrophage subsets that underlie pulmonary pathogenesis in severe COVID-19 and controls the expression of potentially useful biomarkers for COVID-19 severity.
Miriam Simón-Fuentes, Israel Ríos, Cristina Herrero, Fátima Lasala, Nuria Labiod, Joanna Luczkowiak, Emilia Roy-Vallejo, Sara Fernández de Córdoba-Oñate, Pablo Delgado-Wicke, Matilde Bustos, Elena Fernández-Ruiz, Maria Colmenares, Amaya Puig-Kröger, Rafael Delgado, Miguel A. Vega, Ángel L. Corbí, Ángeles Domínguez-Soto
HNF1A haploinsufficiency underlies the most common form of human monogenic diabetes (HNF1A–maturity onset diabetes of the young [HNF1A-MODY]), and hypomorphic HNF1A variants confer type 2 diabetes risk. But a lack of experimental systems for interrogating mature human islets has limited our understanding of how the transcription factor HNF1α regulates adult islet function. Here, we combined conditional genetic targeting in human islet cells, RNA-Seq, chromatin mapping with cleavage under targets and release using nuclease (CUT&RUN), and transplantation-based assays to determine HNF1α-regulated mechanisms in adult human pancreatic α and β cells. Short hairpin RNA–mediated (shRNA-mediated) suppression of HNF1A in primary human pseudoislets led to blunted insulin output and dysregulated glucagon secretion after transplantation in mice, recapitulating phenotypes observed in patients with diabetes. These deficits corresponded with altered expression of genes encoding factors critical for hormone secretion, including calcium channel subunits, ATPase transporters, and extracellular matrix constituents. Additionally, HNF1A loss led to upregulation of transcriptional repressors, providing evidence for a mechanism of transcriptional derepression through HNF1α. CUT&RUN mapping of HNF1α DNA binding sites in primary human islets imputed a subset of HNF1α-regulated genes as direct targets. These data elucidate mechanistic links between HNF1A loss and diabetic phenotypes in mature human α and β cells.
Mollie F. Qian, Romina J. Bevacqua, Vy M.N. Coykendall, Xiong Liu, Weichen Zhao, Charles A. Chang, Xueying Gu, Xiao-Qing Dai, Patrick E. MacDonald, Seung K. Kim
Osteoclasts specialize in bone resorption and are critical for bone remodeling. Previous studies have shown that osteoclasts possess abundant mitochondria and derive most energy through oxidative phosphorylation (OXPHOS). However, the energy substrates fueling OXPHOS in osteoclasts remain to be fully defined. Here, we showed that osteoclast differentiation was coupled with increased oxidation of glucose, glutamine, and oleate. Transcriptomic analyses with RNA sequencing revealed marked upregulation of genes participating in OXPHOS and mitochondrial fatty acid oxidation, during osteoclast differentiation. Increased mitochondrial oxidation of long-chain fatty acids was required for osteoclast differentiation in vitro. However, blocking fatty acid oxidation in vivo, by deletion of carnitine palmitoyltransferase 1a (Cpt1a) in osteoclast progenitors, impaired osteoclast formation only in the female mice. The Cpt1a-deficient females were further protected from osteoclast activation by a high-fat diet. The males, on the contrary, exhibited normal bone resorption despite Cpt1a deletion, regardless of the dietary fat content. Moreover, concurrent deletion of mitochondrial pyruvate carrier 1 and Cpt1a, blocking mitochondrial oxidation of both glucose and fatty acids in the osteoclast lineage, failed to impede bone resorption in the males. The study therefore uncovers a female-specific dependence on mitochondrial oxidation of fatty acids and glucose in osteoclasts in vivo.
Chao Song, Arianna Valeri, Fangfang Song, Xing Ji, Xueyang Liao, Tyler Marmo, Rebecca Seeley, Jared Rutter, Fanxin Long
Idiopathic pulmonary fibrosis (IPF) is a chronic parenchymal lung disease characterized by repetitive alveolar cell injury, myofibroblast proliferation, and excessive extracellular matrix deposition for which unmet need persists for effective therapeutics. The bioactive eicosanoid, prostaglandin F2α, and its cognate receptor FPr (Ptgfr) are implicated as a TGF-β1–independent signaling hub for IPF. To assess this, we leveraged our published murine PF model (IER-SftpcI73T) expressing a disease-associated missense mutation in the surfactant protein C (Sftpc) gene. Tamoxifen-treated IER-SftpcI73T mice developed an early multiphasic alveolitis and transition to spontaneous fibrotic remodeling by 28 days. IER-SftpcI73T mice crossed to a Ptgfr-null (FPr–/–) line showed attenuated weight loss and gene dosage–dependent rescue of mortality compared with FPr+/+ cohorts. IER-SftpcI73T/FPr–/– mice also showed reductions in multiple fibrotic endpoints for which administration of nintedanib was not additive. Single-cell RNA-Seq, pseudotime analysis, and in vitro assays demonstrated Ptgfr expression predominantly within adventitial fibroblasts, which were reprogrammed to an “inflammatory/transitional” cell state in a PGF2α /FPr-dependent manner. Collectively, the findings provide evidence for a role for PGF2α signaling in IPF, mechanistically identify a susceptible fibroblast subpopulation, and establish a benchmark effect size for disruption of this pathway in mitigating fibrotic lung remodeling.
Luis R. Rodriguez, Soon Yew Tang, Willy Roque Barboza, Aditi Murthy, Yaniv Tomer, Tian-Quan Cai, Swati Iyer, Katrina Chavez, Ujjalkumar Subhash Das, Soumita Ghosh, Charlotte H. Cooper, Thalia T. Dimopoulos, Apoorva Babu, Caitlin Connelly, Garret A. FitzGerald, Michael F. Beers
Increased mitochondrial function may render some cancers vulnerable to mitochondrial inhibitors. Since mitochondrial function is regulated partly by mitochondrial DNA copy number (mtDNAcn), accurate measurements of mtDNAcn could help reveal which cancers are driven by increased mitochondrial function and may be candidates for mitochondrial inhibition. However, prior studies have employed bulk macrodissections that fail to account for cell type–specific or tumor cell heterogeneity in mtDNAcn. These studies have often produced unclear results, particularly in prostate cancer. Herein, we developed a multiplex in situ method to spatially quantify cell type–specific mtDNAcn. We show that mtDNAcn is increased in luminal cells of high-grade prostatic intraepithelial neoplasia (HGPIN), is increased in prostatic adenocarcinomas (PCa), and is further elevated in metastatic castration-resistant prostate cancer. Increased PCa mtDNAcn was validated by 2 orthogonal methods and is accompanied by increases in mtRNAs and enzymatic activity. Mechanistically, MYC inhibition in prostate cancer cells decreases mtDNA replication and expression of several mtDNA replication genes, and MYC activation in the mouse prostate leads to increased mtDNA levels in the neoplastic prostate cells. Our in situ approach also revealed elevated mtDNAcn in precancerous lesions of the pancreas and colon/rectum, demonstrating generalization across cancer types using clinical tissue samples.
Jiayu Chen, Qizhi Zheng, Jessica L. Hicks, Levent Trabzonlu, Busra Ozbek, Tracy Jones, Ajay M. Vaghasia, Tatianna C. Larman, Rulin Wang, Mark C. Markowski, Sam R. Denmeade, Kenneth J. Pienta, Ralph H. Hruban, Emmanuel S. Antonarakis, Anuj Gupta, Chi V. Dang, Srinivasan Yegnasubramanian, Angelo M. De Marzo
Control of visceral leishmaniasis (VL) depends on proinflammatory Th1 cells that activate infected tissue macrophages to kill resident intracellular parasites. However, proinflammatory cytokines produced by Th1 cells can damage tissues and require tight regulation. Th1 cell IL-10 production is an important cell–autologous mechanism to prevent such damage. However, IL-10–producing Th1 (type 1 regulatory; Tr1) cells can also delay control of parasites and the generation of immunity following drug treatment or vaccination. To identify molecules to target in order to alter the balance between Th1 and Tr1 cells for improved antiparasitic immunity, we compared the molecular and phenotypic profiles of Th1 and Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6J mice. We also identified a shared Tr1 cell protozoan signature by comparing the transcriptional profiles of Tr1 cells from mice with experimental VL and malaria. We identified LAG3 as an important coinhibitory receptor in patients with VL and experimental VL, and we reveal tissue-specific heterogeneity of coinhibitory receptor expression by Tr1 cells. We also discovered a role for the transcription factor Pbx1 in suppressing CD4+ T cell cytokine production. This work provides insights into the development and function of CD4+ T cells during protozoan parasitic infections and identifies key immunoregulatory molecules.
Chelsea L. Edwards, Jessica A. Engel, Fabian de Labastida Rivera, Susanna S. Ng, Dillon Corvino, Marcela Montes de Oca, Teija C.M. Frame, Shashi Bhushan Chauhan, Siddharth Sankar Singh, Awnish Kumar, Yulin Wang, Jinrui Na, Pam Mukhopadhyay, Jason S. Lee, Susanne Nylen, Shyam Sundar, Rajiv Kumar, Christian R. Engwerda
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