Acute myelogenous leukemia (AML) subtypes that result from oncogenic activation of homeobox (HOX) transcription factors are associated with poor prognosis. The HOXA9 transcription activator and growth factor independent 1 (GFI1) transcriptional repressor compete for occupancy at DNA-binding sites for the regulation of common target genes. We exploited this HOXA9 versus GFI1 antagonism to identify the genes encoding microRNA-21 and microRNA-196b as transcriptional targets of HOX-based leukemia oncoproteins. Therapeutic inhibition of microRNA-21 and microRNA-196b inhibited in vitro leukemic colony forming activity and depleted in vivo leukemia-initiating cell activity of HOX-based leukemias, which led to leukemia-free survival in a murine AML model and delayed disease onset in xenograft models. These data establish microRNA as functional effectors of endogenous HOXA9 and HOX-based leukemia oncoproteins, provide a concise in vivo platform to test RNA therapeutics, and suggest therapeutic value for microRNA antagonists in AML.
Alveolar rhabdomyosarcoma (aRMS) is an aggressive sarcoma of skeletal muscle characterized by expression of the paired box 3-forkhead box protein O1 (
How glucose sensing by the nervous system impacts the regulation of β cell mass and function during postnatal development and throughout adulthood is incompletely understood. Here, we studied mice with inactivation of glucose transporter 2 (
While murine-based systems to identify cancer-promoting agents (carcinogens) are established, models to identify compounds that promote aging (gerontogens) have not been described. For this purpose, we exploited the transcription of
The protective role of hemeoxygenase-1 (HO-1) in various inflammatory conditions is mediated in part by its products, carbon monoxide (CO) and biliverdin. Here we investigated a therapeutic role for CO and CO-primed cells in acute pancreatitis (AP). In a mouse model of AP, treatment with CO-releasing molecule–2 (CORM-2) decreased mortality, pancreatic damage, and lung injury. CORM-2 decreased systemic inflammatory cytokines, suppressed systemic and pancreatic macrophage TNF-α secretion, and inhibited macrophage TLR4 receptor complex expression. In both human and mouse cells, CORM-2 inhibited endogenous and exogenous ligand-dependent TLR4 activation, which indicates that CORM-2 could be therapeutic for both early and late stages of AP, which involve sterile- and endotoxin-mediated inflammation, respectively. Mice engrafted with TLR4-deficient hematopoietic cells were protected against caerulein-induced AP. In the absence of leukocyte TLR4 expression, CORM-2 did not confer additional protection, which indicates that CORM-2–dependent effects are mediated via suppression of macrophage TLR4 activation. We determined that CO was directly responsible for the protective effects of CORM-2 in AP, as inactive forms of CORM-2 were ineffective. Importantly, adoptive transfer of CORM-2–primed cells reduced AP. Such a therapeutic approach would translate the beneficial effects of CO-based therapies, avoiding CO- or CORM-mediated toxicities in AP and a wide range of diseases.
Pancreatic ductal adenocarcinoma (PDAC) is often associated with overexpression of TGF-β. Given its tumor suppressor functions, it is unclear whether TGF-β is a valid therapeutic target for PDAC. Here, we found that proliferating pancreatic cancer cells (PCCs) from human PDAC patients and multiple murine models of PDAC (mPDAC) often exhibit abundant levels of phosphorylated retinoblastoma 1 (RB) and Smad2. TGF-β1 treatment enhanced proliferation of PCCs isolated from
The renal disorder C3 glomerulopathy with dense deposit disease (C3G-DDD) pattern results from complement dysfunction and primarily affects children and young adults. There is no effective treatment, and patients often progress to end-stage renal failure. A small fraction of C3G-DDD cases linked to factor H or C3 gene mutations as well as autoantibodies have been reported. Here, we examined an index family with 2 patients with C3G-DDD and identified a chromosomal deletion in the complement factor H–related (
Lyme disease, caused by the spirochete
There is a considerable resurgence of interest in the role of aerobic glycolysis in cancer; however, increased glycolysis is frequently viewed as a consequence of oncogenic events that drive malignant cell growth and survival. Here we provide evidence that increased glycolytic activation itself can be an oncogenic event in a physiologically relevant 3D culture model. Overexpression of glucose transporter type 3 (GLUT3) in nonmalignant human breast cells activated known oncogenic signaling pathways, including EGFR, β1 integrin, MEK, and AKT, leading to loss of tissue polarity and increased growth. Conversely, reduction of glucose uptake in malignant cells promoted the formation of organized and growth-arrested structures with basal polarity, and suppressed oncogenic pathways. Unexpectedly and importantly, we found that unlike reported literature, in 3D the differences between “normal” and malignant phenotypes could not be explained by HIF-1α/2α, AMPK, or mTOR pathways. Loss of epithelial integrity involved activation of RAP1 via exchange protein directly activated by cAMP (EPAC), involving also O-linked N-acetylglucosamine modification downstream of the hexosamine biosynthetic pathway. The former, in turn, was mediated by pyruvate kinase M2 (PKM2) interaction with soluble adenylyl cyclase. Our findings show that increased glucose uptake activates known oncogenic pathways to induce malignant phenotype, and provide possible targets for diagnosis and therapeutics.
The cytokines RANKL and TNF activate NF-κB signaling in osteoclast precursors (OCPs) to induce osteoclast (OC) formation. Conversely, TNF can limit OC formation through NF-κB p100, which acts as an inhibitor, and TNF receptor–associated receptor 3 (TRAF3); however, a role for TRAF3 in RANKL-mediated OC formation is unknown. We found that TRAF3 limits RANKL-induced osteoclastogenesis by suppressing canonical and noncanonical NF-κB signaling. Conditional OC-specific
The shelterin complex plays dual functions in telomere homeostasis by recruiting telomerase and preventing the activation of a DNA damage response at telomeric ends. Somatic stem cells require telomerase activity, as evidenced by progressive stem cell loss leading to bone marrow failure in hereditary dyskeratosis congenita. Recent work demonstrates that dyskeratosis congenita can also arise from mutations in specific shelterin genes, although little is known about shelterin functions in somatic stem cells. We found that mouse hematopoietic stem cells (HSCs) are acutely sensitive to inactivation of the shelterin gene
Central congenital hypothyroidism (CCH) is more prevalent in children born to women
with hyperthyroidism during pregnancy, suggesting a role for thyroid hormone (TH) in
the development of central thyroid regulation. Using the zebrafish embryo as a model
for thyroid axis development, we have characterized the ontogeny of negative feedback
regulation of thyrotrope function and examined the effect of excess TH on thyrotrope
development. We found that thyroid-stimulating hormone β subunit
Naive T helper cells differentiate into functionally distinct effector subsets that drive specialized immune responses. Recent studies indicate that some of the effector subsets have plasticity. Here, we used an EAE model and found that Th17 cells deficient in the transcription factor BCL11B upregulated the Th2-associated proteins GATA3 and IL-4 without decreasing RAR-related orphan receptor γ (RORγt), IL-17, and GM-CSF levels. Surprisingly, abnormal IL-4 production affected Th17 cell trafficking, diverting migration from the draining lymph nodes/CNS route to the mesenteric lymph nodes/gut route, which ameliorated EAE without overt colitis. T helper cell rerouting in EAE was dependent on IL-4, which enhanced retinoic acid (RA) production by dendritic cells, which further induced expression of gut-homing receptors CCR9 and α4β7 on
Breast cancer (BC) can recur as metastatic disease many years after primary tumor removal, suggesting that disseminated tumor cells survive for extended periods in a dormant state that is refractory to conventional therapies. We have previously shown that altering the tumor microenvironment through fibrosis with collagen and fibronectin deposition can trigger tumor cells to switch from a dormant to a proliferative state. Here, we used an in vivo preclinical model and a 3D in vitro model of dormancy to evaluate the role of Src family kinase (SFK) in regulating this dormant-to-proliferative switch. We found that pharmacological inhibition of SFK signaling or
Metabolic profiling of cancer cells has recently been established as a promising tool for the development of therapies and identification of cancer biomarkers. Here we characterized the metabolomic profile of human breast tumors and uncovered intrinsic metabolite signatures in these tumors using an untargeted discovery approach and validation of key metabolites. The oncometabolite 2-hydroxyglutarate (2HG) accumulated at high levels in a subset of tumors and human breast cancer cell lines. We discovered an association between increased 2HG levels and MYC pathway activation in breast cancer, and further corroborated this relationship using MYC overexpression and knockdown in human mammary epithelial and breast cancer cells. Further analyses revealed globally increased DNA methylation in 2HG-high tumors and identified a tumor subtype with high tissue 2HG and a distinct DNA methylation pattern that was associated with poor prognosis and occurred with higher frequency in African-American patients. Tumors of this subtype had a stem cell–like transcriptional signature and tended to overexpress glutaminase, suggestive of a functional relationship between glutamine and 2HG metabolism in breast cancer. Accordingly, 13C-labeled glutamine was incorporated into 2HG in cells with aberrant 2HG accumulation, whereas pharmacologic and siRNA-mediated glutaminase inhibition reduced 2HG levels. Our findings implicate 2HG as a candidate breast cancer oncometabolite associated with MYC activation and poor prognosis.
Corneal integrity and transparency are indispensable for good vision. Cornea homeostasis is entirely dependent upon corneal stem cells, which are required for complex wound-healing processes that restore corneal integrity following epithelial damage. Here, we found that leucine-rich repeats and immunoglobulin-like domains 1 (
High-dose (HD) IL-2 therapy in patients with cancer increases the general population of Tregs, which are positive for CD4, CD25, and the Treg-specific marker Foxp3. It is unknown whether specific subsets of Tregs are activated and expanded during HD IL-2 therapy or whether activation of any particular Treg subset correlates with clinical outcome. Here, we evaluated Treg population subsets that were induced in patients with melanoma following HD IL-2 therapy. We identified a Treg population that was positive for CD4, CD25, Foxp3, and the inducible T cell costimulator (ICOS). This Treg population increased more than any other lymphocyte subset during HD IL-2 therapy and had an activated Treg phenotype, as indicated by high levels of CD39, CD73, and TGF-β. ICOS+ Tregs were the most proliferative lymphocyte population in the blood after IL-2 therapy. Patients with melanoma with enhanced expansion of ICOS+ Tregs in blood following the first cycle of HD IL-2 therapy had worse clinical outcomes than patients with fewer ICOS+ Tregs. However, there was no difference in total Treg expansion between HD IL-2 responders and nonresponders. These data suggest that increased expansion of the ICOS+ Treg population following the first cycle of HD IL-2 therapy may be predictive of clinical outcome.
Cardiac resynchronization therapy (CRT), the application of biventricular stimulation to correct discoordinate contraction, is the only heart failure treatment that enhances acute and chronic systolic function, increases cardiac work, and reduces mortality. Resting myocyte function also increases after CRT despite only modest improvement in calcium transients, suggesting that CRT may enhance myofilament calcium responsiveness. To test this hypothesis, we examined adult dogs subjected to tachypacing-induced heart failure for 6 weeks, concurrent with ventricular dyssynchrony (HFdys) or CRT. Myofilament force-calcium relationships were measured in skinned trabeculae and/or myocytes. Compared with control, maximal calcium-activated force and calcium sensitivity declined globally in HFdys; however, CRT restored both. Phosphatase PP1 induced calcium desensitization in control and CRT-treated cells, while HFdys cells were unaffected, implying that CRT enhances myofilament phosphorylation. Proteomics revealed phosphorylation sites on Z-disk and M-band proteins, which were predicted to be targets of glycogen synthase kinase-3β (GSK-3β). We found that GSK-3β was deactivated in HFdys and reactivated by CRT. Mass spectrometry of myofilament proteins from HFdys animals incubated with GSK-3β confirmed GSK-3β–dependent phosphorylation at many of the same sites observed with CRT. GSK-3β restored calcium sensitivity in HFdys, but did not affect control or CRT cells. These data indicate that CRT improves calcium responsiveness of myofilaments following HFdys through GSK-3β reactivation, identifying a therapeutic approach to enhancing contractile function.
Recent studies have underscored the importance of memory T cells in mediating protective immunity against pathogens and cancer. Pharmacological inhibition of regulators that mediate T cell differentiation promotes the differentiation of activated CD8+ T cells into memory cells. Nonetheless, pharmacological agents have broad targets and can induce undesirable immunosuppressive effects. Here, we tested the hypothesis that aptamer-targeted siRNA inhibition of mTOR complex 1 (mTORC1) function in CD8+ T cells can enhance their differentiation into memory T cells and potentiate antitumor immunity more effectively than the pharmacologic inhibitor rapamycin. To specifically target activated cells, we conjugated an siRNA targeting the mTORC1 component raptor to an aptamer that binds 4-1BB, a costimulatory molecule that is expressed on CD8+ T cells following TCR stimulation. We found that systemic administration of the 4-1BB aptamer-raptor siRNA to mice downregulated mTORC1 activity in the majority of CD8+ T cells, leading to the generation of a potent memory response that exhibited cytotoxic effector functions and enhanced vaccine-induced protective immunity in tumor-bearing mice. In contrast, while treatment with the general mTORC1 inhibitor rapamycin also enhanced antigen-activated CD8+ T cell persistence, the cytotoxic effector functions of the reactivated memory cells were reduced and the alloreactivity of DCs was diminished. Consistent with the immunological findings, mice treated with rapamycin, but not with 4-1BB aptamer-raptor siRNA, failed to reject a subsequent tumor challenge.
Malaria, which is the result of
Mammals transport blood through a high-pressure, closed vascular network and lymph through a low-pressure, open vascular network. These vascular networks connect at the lymphovenous (LV) junction, where lymph drains into blood and an LV valve (LVV) prevents backflow of blood into lymphatic vessels. Here we describe an essential role for platelets in preventing blood from entering the lymphatic system at the LV junction. Loss of CLEC2, a receptor that activates platelets in response to lymphatic endothelial cells, resulted in backfilling of the lymphatic network with blood from the thoracic duct (TD) in both neonatal and mature mice. Fibrin-containing platelet thrombi were observed at the LVV and in the terminal TD in wild-type mice, but not
The ability of individual T cells to perform multiple effector functions is crucial for protective immunity against viruses and cancer. This polyfunctionality is frequently lost during chronic infections; however, the molecular mechanisms driving T cell polyfunctionality are poorly understood. We found that human T cells stimulated by a high concentration of antigen lacked polyfunctionality and expressed a transcription profile similar to that of exhausted T cells. One specific pathway implicated by the transcription profile in control of T cell polyfunctionality was the MAPK/ERK pathway. This pathway was altered in response to different antigen concentrations, and polyfunctionality correlated with upregulation of phosphorylated ERK. T cells that were stimulated with a high concentration of antigen upregulated sprouty-2 (
Multiple intestinal atresia (MIA) is a rare cause of bowel obstruction that is sometimes associated with a combined immunodeficiency (CID), leading to increased susceptibility to infections. The factors underlying this rare disease are poorly understood. We characterized the immunological and intestinal features of 6 unrelated MIA-CID patients. All patients displayed a profound, generalized lymphocytopenia, with few lymphocytes present in the lymph nodes. The thymus was hypoplastic and exhibited an abnormal distribution of epithelial cells. Patients also had profound disruption of the epithelial barrier along the entire gastrointestinal tract. Using linkage analysis and whole-exome sequencing, we identified 10 mutations in tetratricopeptide repeat domain–7A (
Approximately 85% of lung cancers are non–small-cell lung cancers (NSCLCs), which are often diagnosed at an advanced stage and associated with poor prognosis. Currently, there are very few therapies available for NSCLCs due to the recalcitrant nature of this cancer. Mutations that activate the small GTPase KRAS are found in 20% to 30% of NSCLCs. Here, we report that inhibition of superoxide dismutase 1 (SOD1) by the small molecule ATN-224 induced cell death in various NSCLC cells, including those harboring