Synthetic lethality is an efficient mechanism-based approach to selectively target DNA repair defects. Excision repair cross-complementation group 1 (ERCC1) deficiency is frequently found in non–small-cell lung cancer (NSCLC), making this DNA repair protein an attractive target for exploiting synthetic lethal approaches in the disease. Using unbiased proteomic and metabolic high-throughput profiling on a unique in-house–generated isogenic model of ERCC1 deficiency, we found marked metabolic rewiring of ERCC1-deficient populations, including decreased levels of the metabolite NAD+ and reduced expression of the rate-limiting NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). We also found reduced NAMPT expression in NSCLC samples with low levels of ERCC1. These metabolic alterations were a primary effect of ERCC1 deficiency, and caused selective exquisite sensitivity to small-molecule NAMPT inhibitors, both in vitro — ERCC1-deficient cells being approximately 1,000 times more sensitive than ERCC1-WT cells — and in vivo. Using transmission electronic microscopy and functional metabolic studies, we found that ERCC1-deficient cells harbor mitochondrial defects. We propose a model where NAD+ acts as a regulator of ERCC1-deficient NSCLC cell fitness. These findings open therapeutic opportunities that exploit a yet-undescribed nuclear-mitochondrial synthetic lethal relationship in NSCLC models, and highlight the potential for targeting DNA repair/metabolic crosstalks for cancer therapy.
Mehdi Touat, Tony Sourisseau, Nicolas Dorvault, Roman M. Chabanon, Marlène Garrido, Daphné Morel, Dragomir B. Krastev, Ludovic Bigot, Julien Adam, Jessica R. Frankum, Sylvère Durand, Clement Pontoizeau, Sylvie Souquère, Mei-Shiue Kuo, Sylvie Sauvaigo, Faraz Mardakheh, Alain Sarasin, Ken A. Olaussen, Luc Friboulet, Frédéric Bouillaud, Gérard Pierron, Alan Ashworth, Anne Lombès, Christopher J. Lord, Jean-Charles Soria, Sophie Postel-Vinay
Incidence of nonalcoholic steatohepatitis (NASH), which is considered a hepatic manifestation of metabolic syndrome, has been increasing worldwide with the rise in obesity; however, its pathological mechanism is poorly understood. Here, we demonstrate that the hepatic expression of aortic carboxypeptidase–like protein (ACLP), a glycosylated, secreted protein, increases in NASH in humans and mice. Furthermore, we elucidate that ACLP is a ligand, unrelated to WNT proteins, that activates the canonical WNT pathway and exacerbates NASH pathology. In the liver, ACLP is specifically expressed in hepatic stellate cells (HSCs). As fatty liver disease progresses, ACLP expression is enhanced via activation of STAT3 signaling by obesity-related factors in serum. ACLP specifically binds to frizzled-8 and low-density lipoprotein–related receptor 6 to form a ternary complex that activates canonical WNT signaling. Consequently, ACLP activates HSCs by inhibiting PPARγ signals. HSC-specific ACLP deficiency inhibits fibrosis progression in NASH by inhibiting canonical WNT signaling in HSCs. The present study elucidates the role of canonical WNT pathway activation by ACLP in NASH pathology, indicating that NASH can be treated by targeting ACLP-induced canonical WNT pathway activation in HSCs.
Toshiaki Teratani, Kengo Tomita, Takahiro Suzuki, Hirotaka Furuhashi, Rie Irie, Makoto Nishikawa, Junji Yamamoto, Toshifumi Hibi, Soichiro Miura, Tohru Minamino, Yuichi Oike, Ryota Hokari, Takanori Kanai
Insulin resistance and type 2 diabetes are associated with low levels of high-density lipoprotein cholesterol (HDL-C). The insulin-repressible FoxO transcription factors are potential mediators of the effect of insulin on HDL-C. FoxOs mediate a substantial portion of insulin-regulated transcription, and poor FoxO repression is thought to contribute to the excessive glucose production in diabetes. In this work, we show that mice with liver-specific triple FoxO knockout (L-FoxO1,3,4), which are known to have reduced hepatic glucose production, also have increased HDL-C. This was associated with decreased expression of the HDL-C clearance factors scavenger receptor class B type I (SR-BI) and hepatic lipase and defective selective uptake of HDL cholesteryl ester by the liver. The phenotype could be rescued by re-expression of SR-BI. These findings demonstrate that hepatic FoxOs are required for cholesterol homeostasis and HDL-mediated reverse cholesterol transport to the liver.
Samuel X. Lee, Markus Heine, Christian Schlein, Rajasekhar Ramakrishnan, Jing Liu, Gabriella Belnavis, Ido Haimi, Alexander W. Fischer, Henry N. Ginsberg, Joerg Heeren, Franz Rinninger, Rebecca A. Haeusler
Agonists of the vanilloid receptor transient vanilloid potential 1 (TRPV1) are emerging as highly efficacious nonopioid analgesics in preclinical studies. These drugs selectively lesion TRPV1+ primary sensory afferents, which are responsible for the transmission of many noxious stimulus modalities. Resiniferatoxin (RTX) is a very potent and selective TRPV1 agonist and is a promising candidate for treating many types of pain. Recent work establishing intrathecal application of RTX for the treatment of pain resulting from advanced cancer has demonstrated profound analgesia in client-owned dogs with osteosarcoma. The present study uses transcriptomics and histochemistry to examine the molecular mechanism of RTX action in rats, in clinical canine subjects, and in 1 human subject with advanced cancer treated for pain using intrathecal RTX. In all 3 species, we observe a strong analgesic action, yet this was accompanied by limited transcriptional alterations at the level of the dorsal root ganglion. Functional and neuroanatomical studies demonstrated that intrathecal RTX largely spares susceptible neuronal perikarya, which remain active peripherally but unable to transmit signals to the spinal cord. The results demonstrate that central chemo-axotomy of the TRPV1+ afferents underlies RTX analgesia and refine the neurobiology underlying effective clinical use of TRPV1 agonists for pain control.
Matthew R. Sapio, John K. Neubert, Danielle M. LaPaglia, Dragan Maric, Jason M. Keller, Stephen J. Raithel, Eric L. Rohrs, Ethan M. Anderson, John A. Butman, Robert M. Caudle, Dorothy C. Brown, John D. Heiss, Andrew J. Mannes, Michael J. Iadarola
Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond-Blackfan anemia and 5q– syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor 1 (RNH1) is a ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here, we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 and E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knockdown in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.
Vijaykumar Chennupati, Diogo F.T. Veiga, Kendle M. Maslowski, Nicola Andina, Aubry Tardivel, Eric Chi-Wang Yu, Martina Stilinovic, Cedric Simillion, Michel A. Duchosal, Manfredo Quadroni, Irene Roberts, Vijay G. Sankaran, H. Robson MacDonald, Nicolas Fasel, Anne Angelillo-Scherrer, Pascal Schneider, Trang Hoang, Ramanjaneyulu Allam
Apoptosis has been proposed as a key mechanism responsible for CD4+ T cell depletion and immune dysfunction during HIV infection. We demonstrated that Q-VD-OPH, a caspase inhibitor, inhibits spontaneous and activation-induced death of T cells from SIV-infected rhesus macaques (RMs). When administered during the acute phase of infection, Q-VD-OPH was associated with (a) reduced levels of T cell death, (b) preservation of CD4+/CD8+ T cell ratio in lymphoid organs and in the gut, (c) maintenance of memory CD4+ T cells, and (d) increased specific CD4+ T cell response associated with the expression of cytotoxic molecules. Although therapy was limited to the acute phase of infection, Q-VD-OPH–treated RMs showed lower levels of both viral load and cell-associated SIV DNA as compared with control SIV-infected RMs throughout the chronic phase of infection, and prevented the development of AIDS. Overall, our data demonstrate that Q-VD-OPH injection in SIV-infected RMs may represent an adjunctive therapeutic agent to control HIV infection and delaying disease progression to AIDS.
Mireille Laforge, Ricardo Silvestre, Vasco Rodrigues, Julie Garibal, Laure Campillo-Gimenez, Shahul Mouhamad, Valérie Monceaux, Marie-Christine Cumont, Henintsoa Rabezanahary, Alain Pruvost, Anabela Cordeiro-da-Silva, Bruno Hurtrel, Guido Silvestri, Anna Senik, Jérôme Estaquier
During development, Sox2 is indispensable for cell division and differentiation, yet its roles in regenerating tissues are less clear. Here, we used combinations of transgenic mouse models to reveal that Sox2 haploinsufficiency (Sox2haplo) increases rather than impairs cochlear regeneration in vivo. Sox2haplo cochleae had delayed terminal mitosis and ectopic sensory cells, yet normal auditory function. Sox2haplo amplified and expanded domains of damage-induced Atoh1+ transitional cell formation in neonatal cochlea. Wnt activation via β-catenin stabilization (β-cateninGOF) alone failed to induce proliferation or transitional cell formation. By contrast, β-cateninGOF caused proliferation when either Sox2haplo or damage was present, and transitional cell formation when both were present in neonatal, but not mature, cochlea. Mechanistically, Sox2haplo or damaged neonatal cochleae showed lower levels of Sox2 and Hes5, but not of Wnt target genes. Together, our study unveils an interplay between Sox2 and damage in directing tissue regeneration and Wnt responsiveness and thus provides a foundation for potential combinatorial therapies aimed at stimulating mammalian cochlear regeneration to reverse hearing loss in humans.
Patrick J. Atkinson, Yaodong Dong, Shuping Gu, Wenwen Liu, Elvis Huarcaya Najarro, Tomokatsu Udagawa, Alan G. Cheng
Single cancer cell sequencing studies currently use randomly-selected cells, limiting correlations between genomic aberrations, morphology and spatial localization. We laser-captured microdissected single cells from morphologically-distinct areas of primary breast cancer and corresponding lymph node metastasis and performed whole-exome or deep-target sequencing of greater than 100 such cells. Two major subclones co-existed in different areas of the primary tumor, and the lymph node metastasis originated from a minor subclone in the invasive front of the primary tumor with additional copy number changes including 8q gain, but no additional point mutations in driver genes. Lack of metastasis-specific driver events lead us to assess whether other clonal and subclonal genomic aberrations pre-existing in primary tumors contribute to lymph node metastasis. Gene mutations and copy number variations analyzed in five breast cancer tissue sample sets revealed that copy number variations in several genomic regions, including areas within chromosome 1p, 8q, 9p, 12q and 20q, harboring several metastasis-associated genes, were consistently associated with lymph node metastasis. Moreover, clonal expansion was observed in an area of morphologically-normal breast epithelia, likely driven by a driver mutation and a subsequent amplification in chromosome 1q. Our study illuminates the molecular evolution of breast cancer and genomic aberrations contributing to metastases.
Li Bao, Zhaoyang Qian, Maria B. Lyng, Ling Wang, Yuan Yu, Ting Wang, Xiuqing Zhang, Huanming Yang, Nils Brünner, Jun Wang, Henrik J. Ditzel
Painful diabetic neuropathy (PDN) is an intractable complication of diabetes that affects 25% of patients. PDN is characterized by neuropathic pain and small-fiber degeneration, accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability and loss of their axons within the skin. The molecular mechanisms underlying DRG nociceptor hyperexcitability and small-fiber degeneration in PDN are unknown. We hypothesize that chemokine CXCL12/CXCR4 signaling is central to this mechanism, as we have shown that CXCL12/CXCR4 signaling is necessary for the development of mechanical allodynia, a pain hypersensitivity behavior common in PDN. Focusing on DRG neurons expressing the sodium channel Nav1.8, we applied transgenic, electrophysiological, imaging, and chemogenetic techniques to test this hypothesis. In the high-fat diet mouse model of PDN, we were able to prevent and reverse mechanical allodynia and small-fiber degeneration by limiting CXCR4 signaling or neuronal excitability. This study reveals that excitatory CXCR4/CXCL12 signaling in Nav1.8-positive DRG neurons plays a critical role in the pathogenesis of mechanical allodynia and small-fiber degeneration in a mouse model of PDN. Hence, we propose that targeting CXCR4-mediated DRG nociceptor hyperexcitability is a promising therapeutic approach for disease-modifying treatments for this currently intractable and widespread affliction.
Nirupa D. Jayaraj, Bula J. Bhattacharyya, Abdelhak A. Belmadani, Dongjun Ren, Craig A. Rathwell, Sandra Hackelberg, Brittany E. Hopkins, Herschel R. Gupta, Richard J. Miller, Daniela M. Menichella
Recent studies reveal that airway epithelial cells are critical pulmonary circadian pacemaker cells, mediating rhythmic inflammatory responses. Using mouse models, we now identify the rhythmic circadian repressor REV-ERB as essential to the mechanism coupling the pulmonary clock to innate immunity, involving both myeloid, and bronchial epithelial cells in temporal gating and determining amplitude of response to inhaled endotoxin. Dual mutation of REV-ERBα and its paralog REV-ERBβ in bronchial epithelia further augmented inflammatory responses and chemokine activation, but also initiated a basal inflammatory state, revealing a critical homeostatic role for REV-ERB proteins in the suppression of the endogenous pro-inflammatory mechanism in un-challenged cells. However, REV-ERBα plays the dominant role as deletion of REV-ERBβ alone had no impact on inflammatory responses. In turn, inflammatory challenges cause striking changes in stability and degradation of REV-ERBα protein, driven by SUMOylation and ubiquitination. We developed a novel selective oxazole-based inverse agonist of REV-ERB, which protects REV-ERBα protein from degradation and used this to reveal how pro-inflammatory cytokines trigger rapid degradation of REV-ERα in the elaboration of an inflammatory response. Thus, dynamic changes in stability of REV-ERα protein couple the core clock to innate immunity.
Marie Pariollaud, Julie Gibbs, Thomas Hopwood, Sheila Brown, Nicola Begley, Ryan Vonslow, Toryn Poolman, Baoqiang Guo, Ben Saer, D. Heulyn Jones, James P. Tellam, Stefano Bresciani, Nicholas C.O. Tomkinson, Justyna Wojno-Picon, Anthony W.J. Cooper, Dion A. Daniels, Ryan P. Trump, Daniel Grant, William Zuercher, Timothy M. Willson, Andrew S. MacDonald, Brian Bolognese, Patricia L. Podolin, Yolanda Sanchez, Andrew S.I. Loudon, David W. Ray
Increasing evidence suggests a role for excessive intake of fructose in the Western diet as a contributor to the current epidemics of metabolic syndrome and obesity. Hereditary fructose intolerance (HFI) is a difficult and potentially lethal orphan disease associated with impaired fructose metabolism. In HFI, the deficiency of a particular aldolase, aldolase B, results in the accumulation of intracellular phosphorylated fructose thus leading to phosphate sequestration and depletion, increased ATP turnover and a plethora of conditions leading to clinical manifestations including fatty liver, hyperuricemia, Fanconi syndrome and severe hypoglycemia. Unfortunately, to date, there is no treatment for HFI and avoiding sugar and fructose in our society has become quite challenging. In this report, through use of genetically modified mice and pharmacological inhibitors, we demonstrate that the absence or inhibition of ketohexokinase (Khk), an enzyme upstream of aldolase B, is sufficient to prevent hypoglycemia and liver and intestinal injury associated with HFI using aldolase B knockout mice. We thus provide evidence for the first time of a potential therapeutic approach for this condition. Mechanistically, our studies suggest that it is the inhibition of the Khk C isoform, not the A isoform, that protects animals from HFI.
Miguel A. Lanaspa, Ana Andres-Hernando, David J. Orlicky, Christina Cicerchi, Cholsoon Jang, Nanxing Li, Tamara Milagres, Masanari Kuwabara, Michael F. Wempe, Joshua D. Rabinowitz, Richard J. Johnson, Dean R. Tolan
The remarkable regeneration capability of skeletal muscle depends on coordinated proliferation and differentiation of satellite cells. The self-renewal of satellite cells is critical for long-term maintenance of muscle regeneration potential. Hypoxia profoundly affects the proliferation, differentiation, and self-renewal of cultured myoblasts. However, the physiological relevance of hypoxia and hypoxia signaling in satellite cells in vivo remains largely unknown. Here, we report that satellite cells are in an intrinsic hypoxic state in vivo and express hypoxia-inducible factor 2A (HIF2A). HIF2A promotes the stemness and long-term homeostatic maintenance of satellite cells by maintaining the quiescence, increasing the self-renewal and blocking the myogenic differentiation of satellite cells. HIF2A stabilization in satellite cells cultured under normoxia augmented their engraftment potential in regenerative muscle. Reversely, HIF2A ablation led to the depletion of satellite cells and the consequent regenerative failure in the long-term. In contrast, transient pharmacological inhibition of HIF2A accelerated muscle regeneration by increasing satellite cell proliferation and differentiation. Mechanistically, HIF2A induces the quiescence/self-renewal of satellite cells by binding the promoter of Spry1 gene and activating Spry1 expression. These findings suggest that HIF2A is a pivotal mediator of hypoxia signaling in satellite cells and may be therapeutically targeted to improve muscle regeneration.
Liwei Xie, Amelia Yin, Anna S. Nichenko, Aaron M. Beedle, Jarrod A. Call, Hang Yin
In this issue, Stavrou et al. pursue coagulation factor FXII’s functions in neutrophil-mediated inflammation during wound healing. The cover image depicts translocation of FXII (green) to the plasma membrane of activated neutrophils, where it acts as an autocrine signal to enhance neutrophil adhesion, migration, chemotaxis, and NET formation (purple lines). Image credit: Erika Woodrum.
JCI This Month is a digest of the research, reviews, and other features published each month.
Fibrosis describes a maladaptive response to injury that results in pathogenic production of extracellular matrix, the formation of stiff scar tissue, and compromised organ function. Although it is most often associated with chronic liver conditions and progressive lung disease, fibrosis can affect any organ of the body. There are few treatment options for this progressive, often fatal condition, but as ongoing research identifies the molecular pathways that initiate and propagate fibrotic remodeling, therapeutic possibilities may become available. The reviews in this series discuss recent insights into genetic predisposition to fibrotic disorders, the origins of fibroblasts and myofibroblasts, scar tissue formation, organ regeneration, and more, revealing opportunities to interrupt or even reverse disease progression.