Radiotherapy induces a type I interferon–mediated (T1IFN-mediated) antitumoral immune response that we hypothesized could be potentiated by a first-in-class ataxia telangiectasia mutated (ATM) inhibitor, leading to enhanced innate immune signaling, T1IFN expression, and sensitization to immunotherapy in pancreatic cancer. We evaluated the effects of AZD1390 or a structurally related compound, AZD0156, on innate immune signaling and found that both inhibitors enhanced radiation-induced T1IFN expression via the POLIII/RIG-I/MAVS pathway. In immunocompetent syngeneic mouse models of pancreatic cancer, ATM inhibitor enhanced radiation-induced antitumoral immune responses and sensitized tumors to anti–PD-L1, producing immunogenic memory and durable tumor control. Therapeutic responses were associated with increased intratumoral CD8+ T cell frequency and effector function. Tumor control was dependent on CD8+ T cells, as therapeutic efficacy was blunted in CD8+ T cell–depleted mice. Adaptive immune responses to combination therapy provided systemic control of contralateral tumors outside of the radiation field. Taken together, we show that a clinical candidate ATM inhibitor enhances radiation-induced T1IFN, leading to both innate and subsequent adaptive antitumoral immune responses and sensitization of otherwise resistant pancreatic cancer to immunotherapy.
Qiang Zhang, Long Jiang, Weiwei Wang, Amanda K. Huber, Victoria M. Valvo, Kassidy M. Jungles, Erin A. Holcomb, Ashley N. Pearson, Stephanie The, Zhuwen Wang, Leslie A. Parsels, Joshua D. Parsels, Daniel R. Wahl, Arvind Rao, Vaibhav Sahai, Theodore S. Lawrence, Michael D. Green, Meredith A. Morgan
Fibroblasts are stromal cells known to regulate local immune responses important for wound healing and scar formation; however, the cellular mechanisms driving damage and scarring in patients with cutaneous lupus erythematosus (CLE) remain poorly understood. Dermal fibroblasts in patients with systemic lupus erythematosus (SLE) experience increased cytokine signaling in vivo, but the effect of inflammatory mediators on fibroblast responses in nonscarring versus scarring CLE subtypes is unclear. Here, we examined responses to cytokines in dermal fibroblasts from nonlesional skin of 22 patients with SLE and CLE and 34 individuals acting as healthy controls. Notably, inflammatory cytokine responses were exaggerated in SLE fibroblasts compared with those from individuals acting as healthy controls. In lesional CLE biopsies, these same inflammatory profiles were reflected in single-cell RNA-Seq of SFRP2+ and inflammatory fibroblast subsets, and TGF-β was identified as a critical upstream regulator for inflammatory fibroblasts in scarring discoid lupus lesions. In vitro cytokine stimulation of nonlesional fibroblasts from patients who scar from CLE identified an upregulation of collagens, particularly in response to TGF-β, whereas inflammatory pathways were more prominent in nonscarring patients. Our study revealed that SLE fibroblasts are poised to hyperrespond to inflammation, with differential responses among patients with scarring versus nonscarring disease, providing a potential skin-specific target for mitigating damage.
Suzanne K. Shoffner-Beck, Lisa Abernathy-Close, Stephanie Lazar, Feiyang Ma, Mehrnaz Gharaee-Kermani, Amy Hurst, Craig Dobry, Deepika Pandian, Rachael Wasikowski, Amanda Victory, Kelly Arnold, Johann E. Gudjonsson, Lam C. Tsoi, J. Michelle Kahlenberg
Severe dysfunction in cardiac muscle intracellular Ca2+ handling is a common pathway underlying heart failure. Here we used an inducible genetic model of severe Ca2+ cycling dysfunction by the targeted temporal gene ablation of the cardiac Ca2+ ATPase, SERCA2, in otherwise normal adult mice. In this model, in vivo heart performance was minimally affected initially, even though Serca2a protein was markedly reduced. The mechanism underlying the sustained in vivo heart performance in the weeks prior to complete heart pump failure and death is not clear and is important to understand. Studies were primarily focused on understanding how in vivo diastolic function could be relatively normal under conditions of marked Serca2a deficiency. Interestingly, data show increased cardiac troponin I (cTnI) serine 23/24 phosphorylation content in hearts upon Serca2a ablation in vivo. We report that hearts isolated from the Serca2-deficient mice retained near normal heart pump functional responses to β-adrenergic stimulation. Unexpectedly, using genetic complementation models, in concert with inducible Serca2 ablation, data show that Serca2a-deficient hearts that also lacked the central β-adrenergic signaling–dependent Serca2a negative regulator, phospholamban (PLN), had severe diastolic dysfunction that could still be corrected by β-adrenergic stimulation. Notably, integrating a serines 23/24–to–alanine PKA-refractory sarcomere incorporated cTnI molecular switch complex in mice deficient in Serca2 showed blunting of β-adrenergic stimulation–mediated enhanced diastolic heart performance. Taken together, these data provide evidence of a compensatory regulatory role of the myofilaments as a critical physiological bridging mechanism to aid in preserving heart diastolic performance in failing hearts with severe Ca2+ handling deficits.
Frazer I. Heinis, Brian R. Thompson, Rishi Gulati, Matthew Wheelwright, Joseph M. Metzger
Glucose homeostasis is achieved via complex interactions between the endocrine pancreas and other peripheral tissues and glucoregulatory neurocircuits in the brain that remain incompletely defined. Within the brain, neurons in the hypothalamus appear to play a particularly important role. Consistent with this notion, we report evidence that (pro)renin receptor (PRR) signaling within a subset of tyrosine hydroxylase (TH) neurons located in the hypothalamic paraventricular nucleus (PVNTH neurons) is a physiological determinant of the defended blood glucose level. Specifically, we demonstrate that PRR deletion from PVNTH neurons restores normal glucose homeostasis in mice with diet-induced obesity (DIO). Conversely, chemogenetic inhibition of PVNTH neurons mimics the deleterious effect of DIO on glucose. Combined with our finding that PRR activation inhibits PVNTH neurons, these findings suggest that, in mice, (a) PVNTH neurons play a physiological role in glucose homeostasis, (b) PRR activation impairs glucose homeostasis by inhibiting these neurons, and (c) this mechanism plays a causal role in obesity-associated metabolic impairment.
Shiyue Pan, Lucas A.C. Souza, Caleb J. Worker, Miriam E. Reyes Mendez, Ariana Julia B. Gayban, Silvana G. Cooper, Alfredo Sanchez Solano, Richard N. Bergman, Darko Stefanovski, Gregory J. Morton, Michael W. Schwartz, Yumei Feng Earley
Dedifferentiation or phenotype switching refers to the transition from a proliferative to an invasive cellular state. We previously identified a 122-gene epigenetic gene signature that classifies primary melanomas as low versus high risk (denoted as Epgn1 or Epgn3). We found that the transcriptomes of the Epgn1 low-risk and Epgn3 high-risk cells are similar to the proliferative and invasive cellular states, respectively. These signatures were further validated in melanoma tumor samples. Examination of the chromatin landscape revealed differential H3K27 acetylation in the Epgn1 low-risk versus Epgn3 high-risk cell lines that corroborated with a differential super-enhancer and enhancer landscape. Melanocytic lineage genes (MITF, its targets and regulators) were associated with super-enhancers in the Epgn1 low-risk state, whereas invasiveness genes were linked with Epgn3 high-risk status. We identified the ITGA3 gene as marked by a super-enhancer element in the Epgn3 invasive cells. Silencing of ITGA3 enhanced invasiveness in both in vitro and in vivo systems, suggesting it as a negative regulator of invasion. In conclusion, we define chromatin landscape changes associated with Epgn1/Epgn3 and phenotype switching during early steps of melanoma progression that regulate transcriptional reprogramming. This super-enhancer and enhancer-driven epigenetic regulatory mechanism resulting in major changes in the transcriptome could be important in future therapeutic targeting efforts.
Karen Mendelson, Tiphaine C. Martin, Christie B. Nguyen, Min Hsu, Jia Xu, Claudia Lang, Reinhard Dummer, Yvonne Saenger, Jane L. Messina, Vernon K. Sondak, Garrett Desman, Dan Hasson, Emily Bernstein, Ramon E. Parsons, Julide Tok Celebi
In humans, type 2 diabetes mellitus shows a higher prevalence in men compared with women, a phenotype that has been attributed to a lower peripheral insulin sensitivity in men. Whether sex-specific differences in pancreatic β cell function also contribute is largely unknown. Here, we characterized the electrophysiological properties of β cells in intact male and female mouse islets. Elevation of glucose concentration above 5 mM triggered an electrical activity with a similar glucose dependence in β cells of both sexes. However, female β cells had a more depolarized membrane potential and increased firing frequency compared with males. The higher membrane depolarization in female β cells was caused by approximately 50% smaller Kv2.1 K+ currents compared with males but otherwise unchanged KATP, large-conductance and small-conductance Ca2+-activated K+ channels, and background TASK1/TALK1 K+ current densities. In female β cells, the higher depolarization caused a membrane potential–dependent inactivation of the voltage-gated Ca2+ channels (CaV), resulting in reduced Ca2+ entry. Nevertheless, this reduced Ca2+ influx was offset by a higher action potential firing frequency. Because exocytosis of insulin granules does not show a sex-specific difference, we conclude that the higher electrical activity promotes insulin release in females, improving glucose tolerance.
Noelia Jacobo-Piqueras, Tamara Theiner, Stefanie M. Geisler, Petronel Tuluc
Suppressor of fused (SUFU) is widely regarded as a key negative regulator of the sonic hedgehog (SHH) morphogenic pathway and a known tumor suppressor of medulloblastoma (MB). However, we report here that SUFU expression was markedly increased in 75% of specimens compiled in a tissue array comprising 49 unstratified MBs. The SUFU and GLI1 expression levels in this MB array showed strong positive correlation, which was also identified in a large public data set containing 736 MBs. We further report that increasing Sufu gene dosage in mice caused preaxial polydactyly, which was associated with the expansion of the Gli3 domain in the anterior limb bud and heightened Shh signaling responses during embryonic development. Increasing Sufu gene dosage also led to accelerated cerebellar development and, when combined with ablation of the Shh receptor encoded by Patched1 (Ptch1), promoted MB tumorigenesis. These data reveal multifaceted roles of SUFU in promoting MB tumorigenesis by enhancing SHH signaling. This revelation clarifies potentially counterintuitive clinical observation of high SUFU expression in MBs and may pave way for novel strategies to reduce or reverse MB progression.
Boang Han, Yu Wang, Shen Yue, Yun-hao Zhang, Lun Kuang, Bin-bin Gao, Yue Wang, Ziyu Zhang, Xiaohong Pu, Xin-fa Wang, Chi-chung Hui, Ting-ting Yu, Chen Liu, Steven Y. Cheng
CD4+Foxp3+ regulatory T cells (Tregs) play an essential role in suppressing transplant rejection, but their role within the graft and heterogeneity in tolerance are poorly understood. Here, we compared phenotypic and transcriptomic characteristics of Treg populations within lymphoid organs and grafts in an islet xenotransplant model of tolerance. We showed Tregs were essential for tolerance induction and maintenance. Tregs demonstrated heterogeneity within the graft and lymphoid organs of tolerant mice. A subpopulation of CD127hi Tregs with memory features were found in lymphoid organs, presented in high proportions within long-surviving islet grafts, and had a transcriptomic and phenotypic profile similar to tissue Tregs. Importantly, these memory-like CD127hi Tregs were better able to prevent rejection by effector T cells, after adoptive transfer into secondary Rag–/– hosts, than naive Tregs or unselected Tregs from tolerant mice. Administration of IL-7 to the CD127hi Treg subset was associated with a strong activation of phosphorylation of STAT5. We proposed that memory-like CD127hi Tregs developed within the draining lymph node and underwent further genetic reprogramming within the graft toward a phenotype that had shared characteristics with other tissue or tumor Tregs. These findings suggested that engineering Tregs with these characteristics either in vivo or for adoptive transfer could enhance transplant tolerance.
Yuanfei Zhao, Leigh Nicholson, Hannah Wang, Yi Wen Qian, Wayne J. Hawthorne, Elvira Jimenez-Vera, Brian S. Gloss, Joey Lai, Adwin Thomas, Yi Vee Chew, Heather Burns, Geoff Y. Zhang, Yuan Min Wang, Natasha M. Rogers, Guoping Zheng, Shounan Yi, Stephen I. Alexander, Philip J. O’Connell, Min Hu
Kidney tubules use fatty acid oxidation (FAO) to support their high energetic requirements. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme for FAO, and it is necessary to transport long-chain fatty acids into mitochondria. To define the role of tubular CPT1A in aging and injury, we generated mice with tubule-specific deletion of Cpt1a (Cpt1aCKO mice), and the mice were either aged for 2 years or injured by aristolochic acid or unilateral ureteral obstruction. Surprisingly, Cpt1aCKO mice had no significant differences in kidney function or fibrosis compared with wild-type mice after aging or chronic injury. Primary tubule cells from aged Cpt1aCKO mice had a modest decrease in palmitate oxidation but retained the ability to metabolize long-chain fatty acids. Very-long-chain fatty acids, exclusively oxidized by peroxisomes, were reduced in kidneys lacking tubular CPT1A, consistent with increased peroxisomal activity. Single-nuclear RNA-Seq showed significantly increased expression of peroxisomal FAO enzymes in proximal tubules of mice lacking tubular CPT1A. These data suggest that peroxisomal FAO may compensate in the absence of CPT1A, and future genetic studies are needed to confirm the role of peroxisomal β-oxidation when mitochondrial FAO is impaired.
Safaa Hammoud, Alla Ivanova, Yosuke Osaki, Steven Funk, Haichun Yang, Olga Viquez, Rachel Delgado, Dongliang Lu, Melanie Phillips Mignemi, Jane Tonello, Selene Colon, Louise Lantier, David Wasserman, Benjamin D. Humphreys, Jeffrey Koenitzer, Justin Kern, Mark de Caestecker, Toren Finkel, Agnes Fogo, Nidia Messias, Irfan J. Lodhi, Leslie Gewin
Silver-Russell syndrome (SRS) is a heterogeneous disorder characterized by intrauterine and postnatal growth retardation. HMGA2 variants are a rare cause of SRS and its functional role in human linear growth is unclear. Patients with suspected SRS negative for 11p15LOM/mUPD7 underwent whole-exome and/or targeted-genome sequencing. Mutant HMGA2 protein expression and nuclear localization were assessed. Two Hmga2-knockin mouse models were generated. Five clinical SRS patients harbored HMGA2 variants with differing functional impacts: 2 stop-gain nonsense variants (c.49G>T, c.52C>T), c.166A>G missense variant, and 2 frameshift variants (c.144delC, c.145delA) leading to an identical, extended-length protein. Phenotypic features were highly variable. Nuclear localization was reduced/absent for all variants except c.166A>G. Homozygous knockin mice recapitulating the c.166A>G variant (Hmga2K56E) exhibited a growth-restricted phenotype. An Hmga2Ter76-knockin mouse model lacked detectable full-length Hmga2 protein, similarly to patient 3 and 5 variants. These mice were infertile, with a pygmy phenotype. We report a heterogeneous group of individuals with SRS harboring variants in HMGA2 and describe the first Hmga2 missense knockin mouse model (Hmga2K56E) to our knowledge causing a growth-restricted phenotype. In patients with clinical features of SRS but negative genetic screening, HMGA2 should be included in next-generation sequencing testing approaches.
Avinaash V. Maharaj, Emily Cottrell, Thatchawan Thanasupawat, Sjoerd D. Joustra, Barbara Triggs-Raine, Masanobu Fujimoto, Sarina G. Kant, Danielle van der Kaay, Agnes Clement-de Boers, Alice S. Brooks, Gabriel Amador Aguirre, Irene Martín del Estal, María Inmaculada Castilla de Cortázar Larrea, Ahmed Massoud, Hermine A. van Duyvenvoorde, Christiaan De Bruin, Vivian Hwa, Thomas Klonisch, Sabine Hombach-Klonisch, Helen L. Storr
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