Abstract
Small cell lung cancer (SCLC) is a recalcitrant malignancy with limited treatment options. Bromodomain and extraterminal domain inhibitors (BETis) have shown promising preclinical activity in SCLC, but the broad sensitivity spectrum limits their clinical prospects. Here, we performed unbiased high-throughput drug combination screens to identify therapeutics that could augment the antitumor activities of BETis in SCLC. We found that multiple drugs targeting the PI-3K–AKT–mTOR pathway synergize with BETis, among which mTOR inhibitors (mTORis) show the highest synergy. Using various molecular subtypes of the xenograft models derived from patients with SCLC, we confirmed that mTOR inhibition potentiates the antitumor activities of BETis in vivo without substantially increasing toxicity. Furthermore, BETis induce apoptosis in both in vitro and in vivo SCLC models, and this antitumor effect is further amplified by combining mTOR inhibition. Mechanistically, BETis induce apoptosis in SCLC by activating the intrinsic apoptotic pathway. However, BET inhibition leads to RSK3 upregulation, which promotes survival by activating the TSC2-mTOR-p70S6K1-BAD cascade. mTORis block this protective signaling and augment the apoptosis induced by BET inhibition. Our findings reveal a critical role of RSK3 induction in tumor survival upon BET inhibition and warrant further evaluation of the combination of mTORis and BETis in patients with SCLC.
Authors
Anju Kumari, Lisa Gesumaria, Yan-Jin Liu, V. Keith Hughitt, Xiaohu Zhang, Michele Ceribelli, Kelli M. Wilson, Carleen Klumpp-Thomas, Lu Chen, Crystal McKnight, Zina Itkin, Craig J. Thomas, Beverly A. Mock, David S. Schrump, Haobin Chen
Abstract
Gastrointestinal graft-versus-host disease (GvHD) is a major cause of mortality and morbidity following allogeneic bone marrow transplantation (allo-BMT). Chemerin is a chemotactic protein that recruits leukocytes to inflamed tissues by interacting with ChemR23/CMKLR1, a chemotactic receptor expressed by leukocytes, including macrophages. During acute GvHD, chemerin plasma levels were strongly increased in allo-BM-transplanted mice. The role of the chemerin/CMKLR1 axis in GvHD was investigated using Cmklr1-KO mice. WT mice transplanted with an allogeneic graft from Cmklr1-KO donors (t-KO) had worse survival and more severe GvHD. Histological analysis demonstrated that the gastrointestinal tract was the organ mostly affected by GvHD in t-KO mice. The severe colitis of t-KO mice was characterized by massive neutrophil infiltration and tissue damage associated with bacterial translocation and exacerbated inflammation. Similarly, Cmklr1-KO recipient mice showed increased intestinal pathology in both allogeneic transplant and dextran sulfate sodium–induced colitis. Notably, the adoptive transfer of WT monocytes into t-KO mice mitigated GvHD manifestations by decreasing gut inflammation and T cell activation. In patients, higher chemerin serum levels were predictive of GvHD development. Overall, these results suggest that CMKLR1/chemerin may be a protective pathway for the control of intestinal inflammation and tissue damage in GvHD.
Authors
Erica Dander, Paola Vinci, Stefania Vetrano, Camilla Recordati, Rocco Piazza, Grazia Fazio, Donatella Bardelli, Mattia Bugatti, Francesca Sozio, Andrea Piontini, Sonia Bonanomi, Luca Bertola, Elena Tassistro, Maria Grazia Valsecchi, Stefano Calza, William Vermi, Andrea Biondi, Annalisa Del Prete, Silvano Sozzani, Giovanna D’Amico
Abstract
The adult mammalian heart has limited regenerative capacity, while the neonatal heart fully regenerates during the first week of life. Postnatal regeneration is mainly driven by proliferation of preexisting cardiomyocytes and supported by proregenerative macrophages and angiogenesis. Although the process of regeneration has been well studied in the neonatal mouse, the molecular mechanisms that define the switch between regenerative and nonregenerative cardiomyocytes are not well understood. Here, using in vivo and in vitro approaches, we identified the lncRNA Malat1 as a key player in postnatal cardiac regeneration. Malat1 deletion prevented heart regeneration in mice after myocardial infarction on postnatal day 3 associated with a decline in cardiomyocyte proliferation and reparative angiogenesis. Interestingly, Malat1 deficiency increased cardiomyocyte binucleation even in the absence of cardiac injury. Cardiomyocyte-specific deletion of Malat1 was sufficient to block regeneration, supporting a critical role of Malat1 in regulating cardiomyocyte proliferation and binucleation, a landmark of mature nonregenerative cardiomyocytes. In vitro, Malat1 deficiency induced binucleation and the expression of a maturation gene program. Finally, the loss of hnRNP U, an interaction partner of Malat1, induced similar features in vitro, suggesting that Malat1 regulates cardiomyocyte proliferation and binucleation by hnRNP U to control the regenerative window in the heart.
Authors
Galip S. Aslan, Nicolas Jaé, Yosif Manavski, Youssef Fouani, Mariana Shumliakivska, Lisa Kettenhausen, Luisa Kirchhof, Stefan Günther, Ariane Fischer, Guillermo Luxán, Stefanie Dimmeler
Abstract
Collectin-11 (CL-11) is a recently described soluble C-type lectin that has distinct roles in embryonic development, host defence, autoimmunity, and fibrosis. Here we report that CL-11 also plays an important role in cancer cell proliferation and tumor growth. Melanoma growth was found to be suppressed in Colec11–/– mice in a s.c. B16 melanoma model. Cellular and molecular analyses revealed that CL-11 is essential for melanoma cell proliferation, angiogenesis, establishment of more immunosuppressive tumor microenvironment, and the reprogramming of macrophages to M2 phenotype within melanomas. In vitro analysis revealed that CL-11 can activate tyrosine kinase receptors (EGFR, HER3) and ERK, JNK, and AKT signaling pathways and has a direct stimulatory effect on murine melanoma cell proliferation. Furthermore, blockade of CL-11 (treatment with L-fucose) inhibited melanoma growth in mice. Analysis of open data sets revealed that COLEC11 gene expression is upregulated in human melanomas and that high COLEC11 expression has a trend toward poor survival. CL-11 also had direct stimulatory effects on human tumor cell proliferation in melanoma and several other types of cancer cells in vitro. Overall, our findings provide the first evidence to our knowledge that CL-11 is a key tumor growth–promoting protein and a promising therapeutic target in tumor growth.
Authors
Jia-Xing Wang, Bo Cao, Ning Ma, Kun-Yi Wu, Wan-Bing Chen, Weiju Wu, Xia Dong, Cheng-Fei Liu, Ya-Feng Gao, Teng-Yue Diao, Xiao-Yun Min, Qing Yong, Zong-Fang Li, Wuding Zhou, Ke Li
Abstract
WHIM syndrome is an inherited immune disorder caused by an autosomal dominant heterozygous mutation in CXCR4. The disease is characterized by neutropenia/leukopenia (secondary to retention of mature neutrophils in bone marrow), recurrent bacterial infections, treatment-refractory warts, and hypogammaglobulinemia. All mutations reported in WHIM patients lead to the truncations in the C-terminal domain of CXCR4, R334X being the most frequent. This defect prevents receptor internalization and enhances both calcium mobilization and ERK phosphorylation, resulting in increased chemotaxis in response to the unique ligand CXCL12. Here, we describe 3 patients presenting neutropenia and myelokathexis, but normal lymphocyte count and immunoglobulin levels, carrying what we believe to be a novel Leu317fsX3 mutation in CXCR4, leading to a complete truncation of its intracellular tail. The analysis of the L317fsX3 mutation in cells derived from patients and in vitro cellular models reveals unique signaling features in comparison with R334X mutation. The L317fsX3 mutation impairs CXCR4 downregulation and β-arrestin recruitment in response to CXCL12 and reduces other signaling events — including ERK1/2 phosphorylation, calcium mobilization, and chemotaxis — all processes that are typically enhanced in cells carrying the R334X mutation. Our findings suggest that, overall, the L317fsX3 mutation may be causative of a form of WHIM syndrome not associated with an augmented CXCR4 response to CXCL12.
Authors
Rajesh Kumar, Samantha Milanesi, Martyna Szpakowska, Laura Dotta, Dario Di Silvestre, Anna Maria Trotta, Anna Maria Bello, Mauro Giacomelli, Manuela Benedito, Joana Azevedo, Alexandra Pereira, Emilia Cortesao, Alessandro Vacchini, Alessandra Castagna, Marinella Pinelli, Daniele Moratto, Raffaella Bonecchi, Massimo Locati, Stefania Scala, Andy Chevigné, Elena M. Borroni, Raffaele Badolato
Abstract
Newborns are at high risk of developing neonatal sepsis, particularly if born prematurely. This has been linked to divergent requirements the immune system has to fulfill during intrauterine compared with extrauterine life. By transcriptomic analysis of fetal and adult neutrophils, we shed new light on the molecular mechanisms of neutrophil maturation and functional adaption during fetal ontogeny. We identified an accumulation of differentially regulated genes within the noncanonical NF-κB signaling pathway accompanied by constitutive nuclear localization of RelB and increased surface expression of TNF receptor type II in fetal neutrophils, as well as elevated levels of lymphotoxin α in fetal serum. Furthermore, we found strong upregulation of the negative inflammatory regulator A20 (Tnfaip3) in fetal neutrophils, which was accompanied by pronounced downregulation of the canonical NF-κB pathway. Functionally, overexpressing A20 in Hoxb8 cells led to reduced adhesion of these neutrophil-like cells in a flow chamber system. Conversely, mice with a neutrophil-specific A20 deletion displayed increased inflammation in vivo. Taken together, we have uncovered constitutive activation of the noncanonical NF-κB pathway with concomitant upregulation of A20 in fetal neutrophils. This offers perfect adaption of neutrophil function during intrauterine fetal life but also restricts appropriate immune responses particularly in prematurely born infants.
Authors
Ina Rohwedder, Lou Martha Wackerbarth, Kristina Heinig, Annamaria Ballweg, Johannes Altstätter, Myriam Ripphahn, Claudia Nussbaum, Melanie Salvermoser, Susanne Bierschenk, Tobias Straub, Matthias Gunzer, Marc Schmidt-Supprian, Thomas Kolben, Christian Schulz, Averil Ma, Barbara Walzog, Matthias Heinig, Markus Sperandio
Abstract
Obesity is a major risk factor for end-stage kidney disease. We previously found that lysosomal dysfunction and impaired autophagic flux contribute to lipotoxicity in obesity-related kidney disease, in both humans and experimental animal models. However, the regulatory factors involved in countering renal lipotoxicity are largely unknown. Here, we found that palmitic acid strongly promoted dephosphorylation and nuclear translocation of transcription factor EB (TFEB) by inhibiting the mechanistic target of rapamycin kinase complex 1 pathway in a Rag GTPase–dependent manner, though these effects gradually diminished after extended treatment. We then investigated the role of TFEB in the pathogenesis of obesity-related kidney disease. Proximal tubular epithelial cell–specific (PTEC-specific) Tfeb-deficient mice fed a high-fat diet (HFD) exhibited greater phospholipid accumulation in enlarged lysosomes, which manifested as multilamellar bodies (MLBs). Activated TFEB mediated lysosomal exocytosis of phospholipids, which helped reduce MLB accumulation in PTECs. Furthermore, HFD-fed, PTEC-specific Tfeb-deficient mice showed autophagic stagnation and exacerbated injury upon renal ischemia/reperfusion. Finally, higher body mass index was associated with increased vacuolation and decreased nuclear TFEB in the proximal tubules of patients with chronic kidney disease. These results indicate a critical role of TFEB-mediated lysosomal exocytosis in counteracting renal lipotoxicity.
Authors
Jun Nakamura, Takeshi Yamamoto, Yoshitsugu Takabatake, Tomoko Namba-Hamano, Satoshi Minami, Atsushi Takahashi, Jun Matsuda, Shinsuke Sakai, Hiroaki Yonishi, Shihomi Maeda, Sho Matsui, Isao Matsui, Takayuki Hamano, Masatomo Takahashi, Maiko Goto, Yoshihiro Izumi, Takeshi Bamba, Miwa Sasai, Masahiro Yamamoto, Taiji Matsusaka, Fumio Niimura, Motoko Yanagita, Shuhei Nakamura, Tamotsu Yoshimori, Andrea Ballabio, Yoshitaka Isaka
Abstract
Hypoxia is a sentinel feature of idiopathic pulmonary fibrosis (IPF). The IPF microenvironment contains high lactate levels, and hypoxia enhances cellular lactate production. Lactate, acting through the GPR81 lactate receptor, serves as a signal molecule regulating cellular processes. We previously identified intrinsically fibrogenic mesenchymal progenitor cells (MPCs) that drive fibrosis in the lungs of patients with IPF. However, whether hypoxia enhances IPF MPC fibrogenicity is unclear. We hypothesized that hypoxia increases IPF MPC fibrogenicity via lactate and its cognate receptor GPR81. Here we show that hypoxia promotes IPF MPC self-renewal. The mechanism involves hypoxia-mediated enhancement of LDHA function and lactate production and release. Hypoxia also increases HIF1α levels, and this increase in turn augments the expression of GPR81. Exogenous lactate operating through GPR81 promotes IPF MPC self-renewal. IHC analysis of IPF lung tissue demonstrates IPF MPCs expressing GPR81 and hypoxic markers on the periphery of the fibroblastic focus. We show that hypoxia enhances IPF MPC fibrogenicity in vivo. We demonstrate that knockdown of GPR81 inhibits hypoxia-induced IPF MPC self-renewal in vitro and attenuates hypoxia-induced IPF MPC fibrogenicity in vivo. Our data demonstrate that hypoxia creates a feed-forward loop that augments IPF MPC fibrogenicity via the lactate/GPR81/HIF1α pathway.
Authors
Libang Yang, Adam Gilbertsen, Hong Xia, Alexey Benyumov, Karen Smith, Jeremy Herrera, Emil Racila, Peter B. Bitterman, Craig A. Henke
Abstract
The periodic emergence of SARS-CoV-2 variants of concern (VOCs) with unpredictable clinical severity and ability to escape preexisting immunity emphasizes the continued need for antiviral interventions. Two small molecule inhibitors, molnupiravir (MK-4482), a nucleoside analog, and nirmatrelvir (PF-07321332), a 3C-like protease inhibitor, have recently been approved as monotherapy for use in high-risk patients with COVID-19. As preclinical data are only available for rodent and ferret models, here we assessed the efficacy of MK-4482 and PF-07321332 alone and in combination against infection with the SARS-CoV-2 Delta VOC in the rhesus macaque COVID-19 model. Macaques were infected with the SARS-CoV-2 Delta variant and treated with vehicle, MK-4482, PF-07321332, or a combination of MK-4482 and PF-07321332. Clinical exams were performed at 1, 2, and 4 days postinfection to assess disease and virological parameters. Notably, use of MK-4482 and PF-07321332 in combination improved the individual inhibitory effect of both drugs, resulting in milder disease progression, stronger reduction of virus shedding from mucosal tissues of the upper respiratory tract, stronger reduction of viral replication in the lower respiratory tract, and reduced lung pathology. Our data strongly indicate superiority of combined MK-4482 and PF-07321332 treatment of SARS-CoV-2 infections as demonstrated in the closest COVID-19 surrogate model of human infection.
Authors
Kyle Rosenke, Matt C. Lewis, Friederike Feldmann, Eric Bohrnsen, Benjamin Schwarz, Atsushi Okumura, W. Forrest Bohler, Julie Callison, Carl Shaia, Catharine M. Bosio, Jamie Lovaglio, Greg Saturday, Michael A. Jarvis, Heinz Feldmann
Abstract
Heart failure (HF) is characterized by global alterations in myocardial DNA methylation, yet little is known about the epigenetic regulation of the noncoding genome and potential reversibility of DNA methylation with left ventricular assist device (LVAD) therapy. Genome-wide mapping of myocardial DNA methylation in 36 patients with HF at LVAD implantation, 8 patients at LVAD explantation, and 7 nonfailing (NF) donors using a high-density bead array platform identified 2,079 differentially methylated positions (DMPs) in ischemic cardiomyopathy (ICM) and 261 DMPs in nonischemic cardiomyopathy (NICM). LVAD support resulted in normalization of 3.2% of HF-associated DMPs. Methylation-expression correlation analysis yielded several protein-coding genes that are hypomethylated and upregulated (HTRA1, FBXO16, EFCAB13, and AKAP13) or hypermethylated and downregulated (TBX3) in HF. A potentially novel cardiac-specific super-enhancer long noncoding RNA (lncRNA) (LINC00881) is hypermethylated and downregulated in human HF. LINC00881 is an upstream regulator of sarcomere and calcium channel gene expression including MYH6, CACNA1C, and RYR2. LINC00881 knockdown reduces peak calcium amplitude in the beating human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). These data suggest that HF-associated changes in myocardial DNA methylation within coding and noncoding genomes are minimally reversible with mechanical unloading. Epigenetic reprogramming strategies may be necessary to achieve sustained clinical recovery from heart failure.
Authors
Xianghai Liao, Peter J. Kennel, Bohao Liu, Trevor R. Nash, Richard Z. Zhuang, Amandine F. Godier-Furnemont, Chenyi Xue, Rong Lu, Paolo C. Colombo, Nir Uriel, Muredach P. Reilly, Steven O. Marx, Gordana Vunjak-Novakovic, Veli K. Topkara
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