Inherited germline mutations in the BRCA1 (BReast CAncer gene 1) or BRCA2 (BReast CAncer gene 2) genes (herein BRCA1/2) greatly increase the risk of breast and ovarian cancer, presumably by elevating somatic mutational errors as a consequence of deficient DNA repair. However, this has never been directly demonstrated by a comprehensive analysis of the somatic mutational landscape of primary, non-cancer, mammary epithelial cells of women diagnosed with pathogenic BRCA1 or BRCA2 germline mutations. Here we used an accurate, single-cell whole genome sequencing approach to first show that telomerized primary mammary epithelial cells heterozygous for a highly penetrant BRCA1 variant displayed a robustly elevated mutation frequency as compared to their isogenic control cells. We then demonstrated a small but statistically significant increase in mutation frequency in mammary epithelial cells isolated from the breast of BRCA1/2 mutation carriers as compared to those obtained from age-matched controls with no genetically increased risk for breast cancer.
Shixiang Sun, Kristina Brazhnik, Moonsook Lee, Alexander Y. Maslov, Yi Zhang, Zhenqiu Huang, Susan Klugman, Ben H. Park, Jan Vijg, Cristina Montagna
BACKGROUND. Fasting and NAD+-boosting compounds including NAD+ precursor nicotinamide riboside (NR) confer anti-inflammatory effects. However, the underlying mechanisms and therapeutic potential are incompletely defined. METHODS. We explored the underlying biology in myeloid cells from healthy volunteers following in-vivo placebo or NR administration and subsequently tested the findings in-vitro in monocytes extracted from subjects with systemic lupus erythematosus (SLE). RESULTS. RNA sequencing of unstimulated and lipopolysaccharide (LPS)-activated monocytes implicate NR in the regulation of autophagy and type I interferon signaling. In primary monocytes NR blunts LPS-induced IFNβ production and genetic or pharmacologic disruption of autophagy phenocopies this effect. Given NAD+ is a co-enzyme in oxidoreductive reactions, metabolomics was performed and identified that NR increased inosine level. Inosine supplementation similarly blunts autophagy and IFNβrelease. Finally, as SLE exhibits type I interferon dysregulation, we assessed the NR effect on SLE patient monocytes and found that NR reduces autophagy and interferon-β release. CONCLUSION. We conclude that NR, in an NAD+-dependent manner and in part via inosine-signaling, mediates suppression of autophagy and attenuates type I interferon in myeloid cells and identifies NR as a potential adjunct for SLE management. TRIAL REGISTRATION. ClinicalTrails.gov registration numbers: NCT02812238, NCT00001846 and NCT00001372. FUNDING. This work was supported by the NHLBI and NIAMS Divisions of Intramural Research.
Jing Wu, Komudi Singh, Amy Lin, Allison M. Meadows, Kaiyuan Wu, Vivian Shing, Maximilian Bley, Shahin Hassanzadeh, Rebecca D. Huffstutler, Mark S. Schmidt, Luz P. Blanco, Rong Tian, Charles Brenner, Mehdi Pirooznia, Mariana J. Kaplan, Michael N. Sack
Cancer metastasis is the cause of the majority of cancer-related deaths. In this study, we demonstrated that no/low expression of ATP11B in conjunction with high expression of PTDSS2, which was negatively regulated by BRCA1, markedly accelerates tumor metastasis. Further analysis revealed that low ATP11B-expressing and high PTDSS2-expressing (ATP11Blow/PTDSS2high) cells were associated with poor prognosis and enhanced metastasis in breast cancer patients in general. Mechanistically, an ATP11Blow/PTDSS2high phenotype was associated with increased levels of nonapoptotic phosphatidylserine (PS) on the outer leaflet of the cell membrane. This PS increase serves as a global immunosuppressive signal to promote breast cancer metastasis through an enriched tumor microenvironment with the accumulation of myeloid-derived suppressive cells (MDSCs) and reduced activity of cytotoxic T cells. The metastatic processes associated with ATP11Blow/PTDSSi2hgh cancer cells can be effectively overcome by changing the expression phenotype to ATP11Bhigh/PTDSS2low through a combination of anti-PS antibody with either paclitaxel or docetaxel. Thus, blocking the ATP11Blow/PTDSS2high axis provided a new selective therapeutic strategy to prevent metastasis in breast cancer patients.
Jun Xu, Sek Man Su, Xin Zhang, Un In Chan, Ragini Adhav, Xiaodong Shu, Jianlin Liu, Jianjie Li, Lihua Mo, Yuqing Wang, Tingting An, Josh haipeng Lei, Kai Miao, Chu-Xia Deng, Xiaoling Xu
The bone marrow (BM) microenvironment regulates acute myeloid leukemia (AML) initiation, proliferation and chemotherapy resistance. Following cancer cell death, a growing body of evidence suggests an important role for remaining apoptotic debris in regulating the immunologic response to, and growth of, solid tumors. Here we investigated the role of macrophage LC3-associated phagocytosis (LAP) within the BM microenvironment of AML. Depletion of BM macrophages increased AML growth in-vivo. We showed that LAP is the predominate method of BM macrophage phagocytosis of dead and dying cells in the AML microenvironment. Targeted inhibition of LAP led to accumulation of apoptotic cells (AC) and apoptotic bodies (AB) resulting in accelerated leukemia growth. Mechanistically, LAP of AMLderived-AB by BM macrophages, resulted in STING pathway activation. We identified that AML derived mitochondrial damage associated molecular patterns were processed by BM macrophages via LAP. Moreover, depletion of mitochondrial DNA (mtDNA) in AML derived-AB showed that it is this mtDNA which was responsible for the induction of STING signalling in BM macrophages. Phenotypically we found that STING activation suppressed AML growth through a mechanism related to increased phagocytosis. In summary, we report that macrophage LAP of apoptotic debris in the AML BM microenvironment suppressed tumor growth.
Jamie A. Moore, Jayna J. Mistry, Charlotte Hellmich, Rebecca H. Horton, Edyta E. Wojtowicz, Aisha Jibril, Matthew Jefferson, Thomas Wileman, Naiara Beraza, Kristian M. Bowles, Stuart A. Rushworth
Obstructive arterial diseases including supravalvular aortic stenosis (SVAS), atherosclerosis and restenosis share two important features: an abnormal or disrupted elastic lamellae structure and excessive smooth muscle cells (SMCs). However, the relationship between these pathological features is poorly delineated. SVAS is caused by heterozygous loss-of-function, hypomorphic or deletion mutations in the elastin gene ELN, and SVAS patients and elastin mutant mice display increased arterial wall cellularity and luminal obstructions. Pharmacological treatments for SVAS are lacking as underlying pathobiology is inadequately defined. Herein, using human aortic vascular cells, mouse models as well as aortic samples and SMCs derived from induced pluripotent stem cells of ELN-deficient patients, we demonstrated that elastin insufficiency induced epigenetic changes, upregulating the Notch pathway in SMCs. Specifically, reduced elastin increased levels of γ-secretase, activated NOTCH3 intracellular domain and downstream genes. Notch3 deletion or pharmacological inhibition of γ-secretase attenuated aortic hypermuscularization and stenosis in Eln(-/-) mutants. Eln(-/-) mice expressed higher levels of Notch ligand JAGGED1 (JAG1) in aortic SMCs and endothelial cells (ECs). Finally, Jag1 deletion in SMCs, but not ECs, mitigated the hypermuscular and stenotic phenotype in the aorta of Eln(-/-) mice. Our findings reveal that NOTCH3 pathway upregulation induced pathological aortic SMC accumulation during elastin insufficiency and provide potential therapeutic targets for SVAS.
Jui M. Dave, Raja Chakraborty, Aglaia Ntokou, Junichi Saito, Fatima Z. Saddouk, Zhonghui Feng, Ashish Misra, George Tellides, Robert K. Riemer, Zsolt Urban, Caroline Kinnear, James Ellis, Seema Mital, Robert Mecham, Kathleen A. Martin, Daniel M. Greif
Mutations in TAB2 (transforming growth factor β activated kinase 1 binding protein 2) have been implicated in the pathogenesis of dilated cardiomyopathy and/or congenital heart disease in humans, but the underlying mechanisms are currently unknown. Here we identified an indispensable role for TAB2 in regulating myocardial homeostasis and remodeling by suppressing RIPK1 (receptor-interacting protein kinase 1) activation and RIPK1-dependent apoptosis and necroptosis. Cardiomyocyte-specific deletion of Tab2 in mice triggered dilated cardiomyopathy with massive apoptotic and necroptotic cell death. Moreover, Tab2-deficient mice were also predisposed to myocardial injury and adverse remodeling following pathological stress. In cardiomyocytes, deletion of TAB2, but not its close homologue TAB3, promoted TNFα-induced apoptosis and necroptosis, which was rescued by forced activation of TAK1 or inhibition of RIPK1 kinase activity. Mechanistically, TAB2 critically mediates RIPK1 phosphorylation at Ser321 via a TAK1-dependent mechanism, which prevents RIPK1 kinase activation and the formation of RIPK1-FADD-caspase-8 apoptotic complex or RIPK1-RIPK3 necroptotic complex. Strikingly, genetic inactivation of RIPK1 with Ripk1-K45A knock-in effectively rescued cardiac remodeling and dysfunction in Tab2-deficient mice. Together, these data demonstrate that TAB2 is a key regulator of myocardial homeostasis and remodeling by suppressing RIPK1-dependent apoptosis and necroptosis. Our results also suggest that targeting RIPK1-mediated cell death signaling may represent a promising therapeutic strategy for TAB2 deficiency-induced dilated cardiomyopathy.
Haifeng Yin, Xiaoyun Guo, Yi Chen, Yachang Zeng, Xiaoliang Mo, Siqi Hong, Hui He, Jing Li, Rachel Steinmetz, Qinghang Liu
BACKGROUND. Curative gene therapies for sickle cell disease (SCD) are currently undergoing clinical evaluation. The occurrence of myeloid malignancies in these trials has prompted safety concerns. Individuals with SCD are predisposed to myeloid malignancies, but the underlying causes remain undefined. Clonal hematopoiesis (CH) is a pre-malignant condition that also confers significant predisposition to myeloid cancers. While it has been speculated that CH may play a role in SCD-associated cancer predisposition, limited data addressing this issue have been reported. METHODS. Here, we leveraged 74,190 whole genome sequences to robustly study CH in SCD. Somatic mutation calling methods were used to assess CH in all samples and comparisons between individuals with and without SCD were performed. RESULTS. While we had sufficient power to detect a greater than 2-fold increased rate of CH, we found no detectable variation in rate or clone properties between individuals affected by SCD and controls. The rate of CH in individuals with SCD was unaltered by hydroxyurea use. CONCLUSIONS. We did not observe an increased risk for acquiring detectable CH in SCD, at least as measured by whole genome sequencing. These results should help guide ongoing efforts and further studies that seek to better define the risk factors underlying myeloid malignancy predisposition in SCD and help ensure that curative therapies can be more safely applied.FUNDING. Funding was provided by the New York Stem Cell Foundation and National Institutes of Health. The funders had no role in study design or reporting.
L. Alexander Liggett, Liam D. Cato, Joshua S. Weinstock, Yingze Zhang, S. Mehdi Nouraie, Mark T. Gladwin, Melanie E. Garrett, Allison Ashley-Koch, Marilyn Telen, Brian Custer, Shannon Kelly, Carla Dinardo, Ester C. Sabino, Paula Loureiro, Anna Carneiro-Proietti, Cláudia Maximo, Alexander P. Reiner, Gonçalo R. Abecasis, David A. Williams, Pradeep Natarajan, Alexander G. Bick, Vijay G. Sankaran
The KRAS proto-oncogene is among the most frequently mutated genes in cancer, yet for 40 years it remained an elusive therapeutic target. Recently, allosteric inhibitors that covalently bind to KRAS G12C mutations have been approved for use in lung adenocarcinomas. Although responses are observed they are often short-lived, thus making in-depth characterization of the mechanisms of resistance of paramount importance. Here, we present a rapid-autopsy case of a patient who had a KRASG12C-mutant lung adenocarcinoma who initially responded to a KRAS G12C inhibitor but then rapidly developed resistance. Using deep RNA and whole exome sequencing comparing pre-treatment, post-treatment and matched normal tissues, we uncover numerous mechanisms of resistance to direct KRAS inhibition. In addition to decreased KRAS G12C mutant allele frequency in refractory tumors, we also found reactivation of the MAPK pathway despite no new mutations in KRAS or its downstream mediators. Tumor cell intrinsic and non-cell autonomous mechanisms included increased complement activation, coagulation and tumor angiogenesis, and several lines of evidence of immunologic evasion. Together, our findings reveal numerous mechanisms of resistance to current KRAS G12C inhibitors through enrichment of clonal populations, KRAS-independent downstream signaling and diverse remodeling of the tumor microenvironment.
Yihsuan S. Tsai, Mark G. Woodcock, Salma H. Azam, Leigh B. Thorne, Krishna L. Kanchi, Joel S. Parker, Benjamin G Vincent, Chad V. Pecot
BACKGROUND. Presbyosmia, or aging related olfactory loss, occurs in a majority of humans over age 65 years, yet remains poorly understood, with no specific treatment options. The olfactory epithelium (OE) is the peripheral organ for olfaction, and is subject to acquired damage, suggesting a likely site of pathology in aging. Adult stem cells reconstitute the neuroepithelium in response to cell loss under normal conditions. In aged OE, patches of respiratory-like metaplasia have been observed histologically, consistent with a failure in normal neuroepithelial homeostasis. METHODS. Accordingly, we have focused on identifying cellular and molecular changes in presbyosmic OE. The study combined psychophysical testing with olfactory mucosa biopsy analysis, single cell RNA-sequencing (scRNA-seq), and culture studies. RESULTS. We identified evidence for inflammation-associated changes in the OE stem cells of presbyosmic patients. The presbyosmic basal stem cells exhibited increased expression of genes involved in response to cytokines or stress, or the regulation of proliferation and differentiation. Using a culture model, cytokine exposure drove increased TP63, a transcription factor acting to prevent OE stem cell differentiation. CONCLUSIONS. Our data suggest aging-related inflammatory changes in OE stem cells may contribute to presbyosmia, via the disruption of normal epithelial homeostasis. OE stem cells may represent a therapeutic target for restoration of olfaction. TRIAL REGISTRATION. Not applicable FUNDING. National Institutes of Health grants DC018371 (BJG), NS121067 (EAM), DC016224 (HM);Office of Physician-Scientist Development, Burroughs-Wellcome Fund Research Fellowship for Medical Students Award, Duke University School of Medicine (AO).
Allison D. Oliva, Rupali Gupta, Khalil Issa, Ralph Abi Hachem, David W. Jang, Sebastian A. Wellford, E. Ashley Moseman, Hiroaki Matsunami, Bradley J. Goldstein
Infection with SARS-CoV-2, the causative agent of COVID-19, causes mild to moderate disease in most patients but carries a risk of morbidity and mortality. Seriously affected individuals manifest disorders of hemostasis and a cytokine storm, but it is not understood how these manifestations of severe COVID-19 are linked. Here, we showed that the SARS-CoV-2 Spike protein engaged the CD42b receptor to activate platelet via two distinct signaling pathways, and promoted platelet-monocyte communication through the engagement of P-selectin/PGSL-1 and CD40L/CD40, which led to pro-inflammatory cytokine production by monocytes. These results explain why hypercoagulation, monocyte activation and a cytokine storm are correlated in severely affected COVID-19 patients, and suggest a potential target for therapeutic intervention.
Tianyang Li, Yang Yang, Yongqi Li, Zhengmin Wang, Faxiang Ma, Runqi Luo, Xiaoming Xu, Guo Zhou, Jianhua Wang, Junqi Niu, Guoyue Lv, Ian N Crispe, Zhengkun Tu
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