The transcription factor PU.1 is often impaired in patients with acute myeloid leukemia (AML). Here, we used AML cells that already had low PU.1 levels and further inhibited PU.1 using either RNA interference or, to our knowledge, first-in-class small-molecule inhibitors of PU.1 that we developed specifically to allosterically interfere with PU.1-chromatin binding through interaction with the DNA minor groove that flanks PU.1-binding motifs. These small molecules of the heterocyclic diamidine family disrupted the interaction of PU.1 with target gene promoters and led to downregulation of canonical PU.1 transcriptional targets. shRNA or small-molecule inhibition of PU.1 in AML cells from either PU.1lo mutant mice or human patients with AML-inhibited cell growth and clonogenicity and induced apoptosis. In murine and human AML (xeno)transplantation models, treatment with our PU.1 inhibitors decreased tumor burden and resulted in increased survival. Thus, our study provides proof of concept that PU.1 inhibition has potential as a therapeutic strategy for the treatment of AML and for the development of small-molecule inhibitors of PU.1.
Iléana Antony-Debré, Ananya Paul, Joana Leite, Kelly Mitchell, Hye Mi Kim, Luis A. Carvajal, Tihomira I. Todorova, Kenneth Huang, Arvind Kumar, Abdelbasset A. Farahat, Boris Bartholdy, Swathi-Rao Narayanagari, Jiahao Chen, Alberto Ambesi-Impiombato, Adolfo A. Ferrando, Ioannis Mantzaris, Evripidis Gavathiotis, Amit Verma, Britta Will, David W. Boykin, W. David Wilson, Gregory M.K. Poon, Ulrich Steidl
Deficiency in Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3) in humans is associated with the development of congenital hypothyroidism. However, the functions of GLIS3 in the thyroid gland and the mechanism by which GLIS3 dysfunction causes hypothyroidism are unknown. In the current study, we demonstrate that GLIS3 acts downstream of thyroid-stimulating hormone (TSH) and TSH receptor (TSHR) and is indispensable for TSH/TSHR-mediated proliferation of thyroid follicular cells and biosynthesis of thyroid hormone. Using ChIP-Seq and promoter analysis, we demonstrate that GLIS3 is critical for the transcriptional activation of several genes required for thyroid hormone biosynthesis, including the iodide transporters Nis and Pds, both of which showed enhanced GLIS3 binding at their promoters. The repression of cell proliferation of GLIS3-deficient thyroid follicular cells was due to the inhibition of TSH-mediated activation of the mTOR complex 1/ribosomal protein S6 (mTORC1/RPS6) pathway as well as the reduced expression of several cell division–related genes regulated directly by GLIS3. Consequently, GLIS3 deficiency in a murine model prevented the development of goiter as well as the induction of inflammatory and fibrotic genes during chronic elevation of circulating TSH. Our study identifies GLIS3 as a key regulator of TSH/TSHR-mediated thyroid hormone biosynthesis and proliferation of thyroid follicular cells and uncovers a mechanism by which GLIS3 deficiency causes neonatal hypothyroidism and prevents goiter development.
Hong Soon Kang, Dhirendra Kumar, Grace Liao, Kristin Lichti-Kaiser, Kevin Gerrish, Xiao-Hui Liao, Samuel Refetoff, Raja Jothi, Anton M. Jetten
Attention-deficit/hyperactivity disorder (ADHD) is a prevalent psychiatric disorder in children. Although an imbalance of excitatory and inhibitory inputs has been proposed as contributing to this disorder, the mechanisms underlying this highly heterogeneous disease remain largely unknown. Here, we show that N-myc downstream-regulated gene 2 (NDRG2) deficiency is involved in the development of ADHD in both mice and humans. Ndrg2-knockout (Ndrg2–/–) mice exhibited ADHD-like symptoms characterized by attention deficits, hyperactivity, impulsivity, and impaired memory. Furthermore, interstitial glutamate levels and excitatory transmission were markedly increased in the brains of Ndrg2–/– mice due to reduced astroglial glutamate clearance. We developed an NDRG2 peptide that rescued astroglial glutamate clearance and reduced excitatory glutamate transmission in NDRG2-deficient astrocytes. Additionally, NDRG2 peptide treatment rescued ADHD-like hyperactivity in the Ndrg2–/– mice, while routine methylphenidate treatment had no effect on hyperactivity in these animals. Finally, children who were heterozygous for rs1998848, a SNP in NDRG2, had a higher risk of ADHD than children who were homozygous for rs1998848. Our results indicate that NDRG2 deficiency leads to ADHD phenotypes and that impaired astroglial glutamate clearance, a mechanism distinct from the well-established dopamine deficit hypothesis for ADHD, underlies the resultant behavioral abnormalities.
Yan Li, Anqi Yin, Xin Sun, Ming Zhang, Jianfang Zhang, Ping Wang, Rougang Xie, Wen Li, Ze Fan, Yuanyuan Zhu, Han Wang, Hailong Dong, Shengxi Wu, Lize Xiong
Molybdenum cofactor deficiency (MoCD) is an autosomal recessive inborn error of metabolism characterized by neurodegeneration and death in early childhood. The rapid and progressive neurodegeneration in MoCD presents a major clinical challenge and may relate to the poor understanding of the molecular mechanisms involved. Recently, we reported that treating patients with cyclic pyranopterin monophosphate (cPMP) is a successful therapy for a subset of infants with MoCD and prevents irreversible brain damage. Here, we studied S-sulfocysteine (SSC), a structural analog of glutamate that accumulates in the plasma and urine of patients with MoCD, and demonstrated that it acts as an N-methyl D-aspartate receptor (NMDA-R) agonist, leading to calcium influx and downstream cell signaling events and neurotoxicity. SSC treatment activated the protease calpain, and calpain-dependent degradation of the inhibitory synaptic protein gephyrin subsequently exacerbated SSC-mediated excitotoxicity and promoted loss of GABAergic synapses. Pharmacological blockade of NMDA-R, calcium influx, or calpain activity abolished SSC and glutamate neurotoxicity in primary murine neurons. Finally, the NMDA-R antagonist memantine was protective against the manifestation of symptoms in a tungstate-induced MoCD mouse model. These findings demonstrate that SSC drives excitotoxic neurodegeneration in MoCD and introduce NMDA-R antagonists as potential therapeutics for this fatal disease.
Avadh Kumar, Borislav Dejanovic, Florian Hetsch, Marcus Semtner, Debora Fusca, Sita Arjune, Jose Angel Santamaria-Araujo, Aline Winkelmann, Scott Ayton, Ashley I. Bush, Peter Kloppenburg, Jochen C. Meier, Guenter Schwarz, Abdel Ali Belaidi
Synovial sarcoma (SS) is an aggressive soft-tissue sarcoma that is often discovered during adolescence and young adulthood. Despite the name, synovial sarcoma does not typically arise from a synoviocyte but instead arises in close proximity to bones. Previous work demonstrated that mice expressing the characteristic SS18-SSX fusion oncogene in myogenic factor 5–expressing (Myf5-expressing) cells develop fully penetrant sarcomagenesis, suggesting skeletal muscle progenitor cell origin. However, Myf5 is not restricted to committed myoblasts in embryos but is also expressed in multipotent mesenchymal progenitors. Here, we demonstrated that human SS and mouse tumors arising from SS18-SSX expression in the embryonic, but not postnatal, Myf5 lineage share an anatomic location that is frequently adjacent to bone. Additionally, we showed that SS can originate from periosteal cells expressing SS18-SSX alone and from preosteoblasts expressing the fusion oncogene accompanied by the added stabilization of β-catenin, which is a common secondary change in SS. Expression and secretion of the osteoclastogenesis inhibitory factor osteoprotegerin enabled early growth of SS18-SSX2–transformed cells, indicating a paracrine link between the bone and synovial sarcomagenesis. These findings explain the skeletal contact frequently observed in human SS and may provide alternate means of enabling SS18-SSX–driven oncogenesis in cells as differentiated as preosteoblasts.
Jared J. Barrott, Benjamin E. Illum, Huifeng Jin, Matthew L. Hedberg, Yanliang Wang, Allie Grossmann, Malay Haldar, Mario R. Capecchi, Kevin B. Jones
Retinitis pigmentosa (RP) is a major cause of blindness that affects 1.5 million people worldwide. Mutations in cyclic nucleotide-gated channel β 1 (CNGB1) cause approximately 4% of autosomal recessive RP. Gene augmentation therapy shows promise for treating inherited retinal degenerations; however, relevant animal models and biomarkers of progression in patients with RP are needed to assess therapeutic outcomes. Here, we evaluated RP patients with CNGB1 mutations for potential biomarkers of progression and compared human phenotypes with those of mouse and dog models of the disease. Additionally, we used gene augmentation therapy in a CNGβ1-deficient dog model to evaluate potential translation to patients. CNGB1-deficient RP patients and mouse and dog models had a similar phenotype characterized by early loss of rod function and slow rod photoreceptor loss with a secondary decline in cone function. Advanced imaging showed promise for evaluating RP progression in human patients, and gene augmentation using adeno-associated virus vectors robustly sustained the rescue of rod function and preserved retinal structure in the dog model. Together, our results reveal an early loss of rod function in CNGB1-deficient patients and a wide window for therapeutic intervention. Moreover, the identification of potential biomarkers of outcome measures, availability of relevant animal models, and robust functional rescue from gene augmentation therapy support future work to move CNGB1-RP therapies toward clinical trials.
Simon M. Petersen-Jones, Laurence M. Occelli, Paige A. Winkler, Winston Lee, Janet R. Sparrow, Mai Tsukikawa, Sanford L. Boye, Vince Chiodo, Jenina E. Capasso, Elvir Becirovic, Christian Schön, Mathias W. Seeliger, Alex V. Levin, Stylianos Michalakis, William W. Hauswirth, Stephen H. Tsang
Medulloblastoma, an aggressive cancer of the cerebellum, is among the most common pediatric brain tumors. Approximately one-third of medulloblastomas are associated with misactivation of the Hedgehog (Hh) pathway. GLI family zinc finger 2 (GLI2) coordinates the Hh transcriptional program; however, the GLI2 targets that promote cancer cell proliferation are unknown. Here, we incorporated a Gli2-EGFP allele into 2 different genetic mouse models of Hh-associated medulloblastoma. Hh signaling induced GLI2 binding to the Cdk6 promoter and activated Cdk6 expression, thereby promoting uncontrolled cell proliferation. Genetic or pharmacological inhibition of CDK6 in mice repressed the growth of Hh-associated medulloblastoma and prolonged survival through inhibition of cell proliferation. In human medulloblastoma, misactivation of Hh signaling was associated with high levels of CDK6, pointing to CDK6 as a direct transcriptional target of the Hh pathway. These results suggest that CDK6 antagonists may be a promising therapeutic approach for Hh-associated medulloblastoma in humans.
David R. Raleigh, Pervinder K. Choksi, Alexis Leigh Krup, Wasima Mayer, Nicole Santos, Jeremy F. Reiter
The incorporation of excess saturated free fatty acids (SFAs) into membrane phospholipids within the ER promotes ER stress, insulin resistance, and hepatic gluconeogenesis. Thioesterase superfamily member 2 (Them2) is a mitochondria-associated long-chain fatty acyl-CoA thioesterase that is activated upon binding phosphatidylcholine transfer protein (PC-TP). Under fasting conditions, the Them2/PC-TP complex directs saturated fatty acyl-CoA toward β-oxidation. Here, we showed that during either chronic overnutrition or acute induction of ER stress, Them2 and PC-TP play critical roles in trafficking SFAs into the glycerolipid biosynthetic pathway to form saturated phospholipids, which ultimately reduce ER membrane fluidity. The Them2/PC-TP complex activated ER stress pathways by enhancing translocon-mediated efflux of ER calcium. The increased cytosolic calcium, in turn, led to the phosphorylation of calcium/calmodulin-dependent protein kinase II, which promoted both hepatic insulin resistance and gluconeogenesis. These findings delineate a mechanistic link between obesity and insulin resistance and establish the Them2/PC-TP complex as an attractive target for the management of hepatic steatosis and insulin resistance.
Baran A. Ersoy, Kristal M. Maner-Smith, Yingxia Li, Ipek Alpertunga, David E. Cohen
V617F driver mutation of JAK2 is the leading cause of the Philadelphia-chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombosis is a leading cause of mortality and morbidity in MPNs, the mechanisms underlying their pathogenesis are unclear. Here, we identified pleckstrin-2 (Plek2) as a downstream target of the JAK2/STAT5 pathway in erythroid and myeloid cells, and showed that it is upregulated in a JAK2V617F-positive MPN mouse model and in patients with MPNs. Loss of Plek2 ameliorated JAK2V617F-induced myeloproliferative phenotypes including erythrocytosis, neutrophilia, thrombocytosis, and splenomegaly, thereby reverting the widespread vascular occlusions and lethality in JAK2V617F-knockin mice. Additionally, we demonstrated that a reduction in red blood cell mass was the main contributing factor in the reversion of vascular occlusions. Thus, our study identifies Plek2 as an effector of the JAK2/STAT5 pathway and a key factor in the pathogenesis of JAK2V617F-induced MPNs, pointing to Plek2 as a viable target for the treatment of MPNs.
Baobing Zhao, Yang Mei, Lan Cao, Jingxin Zhang, Ronen Sumagin, Jing Yang, Juehua Gao, Matthew J. Schipma, Yanfeng Wang, Chelsea Thorsheim, Liang Zhao, Timothy Stalker, Brady Stein, Qiang Jeremy Wen, John D. Crispino, Charles S. Abrams, Peng Ji
Humoral rejection is the most common cause of solid organ transplant failure. Here, we evaluated a cohort of 49 patients who were successfully grafted with allogenic islets and determined that the appearance of donor-specific anti-HLA antibodies (DSAs) did not accelerate the rate of islet graft attrition, suggesting resistance to humoral rejection. Murine DSAs bound to allogeneic targets expressed by islet cells and induced their destruction in vitro; however, passive transfer of the same DSAs did not affect islet graft survival in murine models. Live imaging revealed that DSAs were sequestrated in the circulation of the recipients and failed to reach the endocrine cells of grafted islets. We used murine heart transplantation models to confirm that endothelial cells were the only accessible targets for DSAs, which induced the development of typical microvascular lesions in allogeneic transplants. In contrast, the vasculature of DSA-exposed allogeneic islet grafts was devoid of lesions because sprouting of recipient capillaries reestablished blood flow in grafted islets. Thus, we conclude that endothelial chimerism combined with vascular sequestration of DSAs protects islet grafts from humoral rejection. The reduced immunoglobulin concentrations in the interstitial tissue, confirmed in patients, may have important implications for biotherapies such as vaccines and monoclonal antibodies.
Chien-Chia Chen, Eric Pouliquen, Alexis Broisat, Francesco Andreata, Maud Racapé, Patrick Bruneval, Laurence Kessler, Mitra Ahmadi, Sandrine Bacot, Carole Saison-Delaplace, Marina Marcaud, Jean-Paul Duong Van Huyen, Alexandre Loupy, Jean Villard, Sandrine Demuylder-Mischler, Thierry Berney, Emmanuel Morelon, Meng-Kun Tsai, Marie-Nathalie Kolopp-Sarda, Alice Koenig, Virginie Mathias, Stéphanie Ducreux, Catherine Ghezzi, Valerie Dubois, Antonino Nicoletti, Thierry Defrance, Olivier Thaunat
In this month’s JCI, Poulos et al. reveal that the age of bone marrow endothelial cells (BMECs) strongly influences hematopoietic stem cell characteristics. Whereas aged endothelium imparted an aging phenotype to young hematopoietic stem cells, young endothelium rescued many of the deficits observed in aged cells. In the cover image, upregulation of ROS (magenta) represents one age-related defect observed in older BMECs (visualized in red and green with blue nuclei). Image credit: Michael Gutkin.
JCI This Month is a digest of the research, reviews, and other features published each month.
Glia are central nervous system cells that surround neurons and hold them in place, supply them with nutrients and oxygen, serve to insulate neurons from each other, and to remove pathogens and dead neurons. Historically, these cells have been considered less interesting than neurons; however, in the past decade, they have emerged as critical regulators of brain development and homeostasis and are now being appreciated as drivers of disease. Reviews in this series describe the role of glia and the associated glymphatic system in normal physiology, neuronal metabolism, prion diseases, Alzheimer’s disease, ALS, spinal cord injury, and neurodegenerative disease.