Humanized neurofibroma model from induced pluripotent stem cells delineates tumor pathogenesis and developmental origins

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Abstract

Neurofibromatosis type 1 (NF1) is a common tumor predisposition syndrome, caused by NF1 gene mutation, in which affected patients develop Schwann cell lineage peripheral nerve sheath tumors (neurofibromas). To investigate human neurofibroma pathogenesis, we differentiated a series of isogenic patient-specific NF1-mutant human induced-pluripotent stem cells (hiPSCs) into Schwannian lineage cells (SLCs). We found that while wild-type and heterozygous NF1-mutant hiPSC-SLCs did not form tumors following mouse sciatic nerve implantation, NF1-null SLCs formed bona fide neurofibromas with high levels of SOX10 expression. To confirm that SOX10+ SLCs contain the cells of origin for neurofibromas, both Nf1 alleles were inactivated in mouse Sox10+ cells, leading to classic nodular cutaneous and plexiform neurofibroma formation that completely recapitulate their human counterparts. Moreover, we discovered that NF1 loss impaired Schwann cell differentiation by inducing a persistent stem-like state to expand the pool of progenitors required to initiate tumor formation, indicating that in addition to regulating MAPK-mediated cell growth, NF1 loss also alters Schwann cell differentiation to promote neurofibroma development. Taken together, we established complementary humanized neurofibroma explant and first-in-kind mouse genetically engineered nodular cutaneous neurofibroma models that delineate neurofibroma pathogenesis amenable to future therapeutic target discovery and evaluation.

Authors

Juan Mo, Corina Anastasaki, Zhiguo Chen, Tracey Shipman, Jason B. Papke, Kevin Y. Yin, David H. Gutmann, Lu Q. Le

Neutrophilic inflammation during lung development disrupts elastin assembly and predisposes adult mice to COPD

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Abstract

Emerging evidence indicates that early life events can increase the risk for developing chronic obstructive pulmonary disease (COPD). Using an inducible transgenic mouse model for NF-κB activation in the airway epithelium, we found that a brief period of inflammation during the saccular stage [postnatal day (PN)3 - PN5] but not alveolar stage (PN10 - PN12) of lung development disrupts elastic fiber assembly, resulting in permanent reduction in lung function and development of a COPD-like lung phenotype that progresses through 24 months of age. Neutrophil depletion prevented disruption of elastic fiber assembly and restored normal lung development. Mechanistic studies uncovered a role for neutrophil elastase (NE) in downregulating expression of critical elastic fiber assembly components, particularly fibulin-5 and elastin. Further, both purified human NE and NE-containing exosomes from tracheal aspirates of premature infants with lung inflammation down-regulated elastin and fibulin-5 expression by saccular stage mouse lung fibroblasts. Together, our studies define a critical developmental window for assembling the elastin scaffold in the distal lung, which is required to support lung structure and function throughout the lifespan. While neutrophils play a well-recognized role in COPD development in adults, neutrophilic inflammation may also contribute to early life predisposition to COPD.

Authors

John T. Benjamin, Erin Plosa, Jennifer Sucre, Riet van der Meer, Shivangi Dave, Sergey S. Gutor, David Nichols, Peter Gulleman, Christopher Jetter, Wei Han, Matthew K. Xin, Peter C. Dinella, Ashley Catanzarite, Seunghyi Kook, Kalsang Dolma, Charitharth V. Lal, Amit Gaggar, J. Edwin Blalock, Dawn C. Newcomb, Bradley W. Richmond, Jonathan A. Kropski, Lisa R. Young, Susan Guttentag, Timothy S. Blackwell

Graft-versus-host disease depletes plasmacytoid dendritic cell progenitors to impair tolerance induction

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Abstract

Graft-versus-host disease (GVHD) causes failed reconstitution of donor plasmacytoid dendritic cells (pDCs) that are critical for immune protection and tolerance. We used both murine and human systems to uncover the mechanisms whereby GVHD induces donor pDC defects. GVHD depleted Flt3-expressing donor multipotent progenitors (MPPs) that sustained pDCs, leading to impaired generation of pDCs. MPP loss was associated with decreased amounts of MPP-producing hematopoietic stem cells (HSCs) and oxidative stress-induced death of proliferating MPPs. Additionally, alloreactive T cells produced GM-CSF to inhibit MPP expression of Tcf4, the transcription factor essential for pDC development, subverting MPP production of pDCs. GM-CSF did not affect the maturation of pDC precursors. Notably, enhanced recovery of donor pDCs upon adoptive transfer early after allogeneic HSC transplantation repressed GVHD and restored the de novo generation of donor pDCs in recipient mice. pDCs suppressed the proliferation and expansion of activated autologous T cells via a type-I IFN signaling-dependent mechanism. They also produced PD-L1 and LILRB4 to inhibit T cell production of IFN-. We thus demonstrate that GVHD impairs the reconstitution of tolerogenic donor pDCs by depleting DC progenitors rather than by preventing pDC maturation. MPPs are an important target to effectively bolster pDC reconstitution for controlling GVHD.

Authors

Yuanyuan Tian, Lijun Meng, Ying Wang, Bohan Li, Hongshuang Yu, Yan Zhou, Tien Bui, Alicia Li, Ciril Abraham, Yongping Zhang, Jian Wang, Chenchen Zhao, Shin Mineishi, Stefania Gallucci, David Porter, Elizabeth Hexner, Hong Zheng, Yanyun Zhang, Shaoyan Hu, Yi Zhang

CLIC1 recruits PIP5K1A/C to induce cell-matrix adhesions for tumor metastasis

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Abstract

Membrane protrusion and adhesion to the extracellular matrix, which involves the extension of actin filaments and formation of adhesion complexes, are the fundamental processes for cell migration, tumor invasion, and metastasis. How cancer cells efficiently coordinate these processes remains unclear. Here, we showed that membrane-targeted CLIC1 spatiotemporally regulates the formation of cell-matrix adhesions and membrane protrusions through the recruitment of PIP5Ks to the plasma membrane. Comparative proteomics identified CLIC1 upregulated in human hepatocellular carcinoma (HCC) and associated with tumor invasiveness, metastasis, and poor prognosis. In response to migration-related stimuli, CLIC1 recruited PIP5K1A and PIP5K1C from the cytoplasm to the leading edge of the plasma membrane, where PIP5Ks generate a PIP2-rich microdomain to induce the formation of integrin-mediated cell-matrix adhesions and the signaling for cytoskeleon extension. CLIC1 silencing inhibited the attachment of tumor cells to culture plates and the adherence and extravasation in the lung alveoli resulting in suppressed lung metastasis in mice. This study reveals an unrecognized mechanism that spatiotemporally coordinates the formation of both lamellipodium/invadopodia and nascent cell-matrix adhesions for directional migration and tumor invasion/metastasis. The unique traits of upregulation and membrane targeting of CLIC1 in cancer cells make it an excellent therapeutic target for tumor metastasis.

Authors

Jei-Ming Peng, Sheng-Hsuan Lin, Ming-Chin Yu, Sen-Yung Hsieh

Guanosine triphosphate links MYC-dependent metabolic and ribosome programs in small cell lung cancer

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Abstract

MYC stimulates both metabolism and protein synthesis, but it is unknown how cells coordinate these complementary programs. Previous work reported that in a subset of small cell lung cancer (SCLC) cell lines, MYC activates guanosine triphosphate (GTP) synthesis and results in sensitivity to inhibitors of the GTP synthesis enzyme inosine monophosphate dehydrogenase (IMPDH). Here we demonstrated that primary MYCHigh human SCLC tumors also contain abundant guanosine nucleotides. We also found that elevated MYC in SCLCs with acquired chemoresistance rendered these otherwise recalcitrant tumors dependent on IMPDH. Unexpectedly, our data indicated that IMPDH links the metabolic and protein synthesis outputs of oncogenic MYC. Co-expression analysis placed IMPDH within the MYC-driven ribosome program, and GTP depletion prevented RNA Polymerase I (Pol I) from localizing to ribosomal DNA. Furthermore, the GTPases GPN1 and GPN3 were upregulated by MYC and directed Pol I to ribosomal DNA. Constitutively GTP-bound GPN1/3 mutants mitigated the effect of GTP depletion on Pol I, protecting chemoresistant SCLC cells from IMPDH inhibition. GTP therefore functions as a metabolic gate tethering MYC-dependent ribosome biogenesis to nucleotide sufficiency through GPN1 and GPN3. IMPDH dependence is a targetable vulnerability in chemoresistant, MYCHigh SCLC.

Authors

Fang Huang, Kenneth Huffman, Zixi Wang, Xun Wang, Kailong Li, Feng Cai, Chendong Yang, Ling Cai, Terry S. Shih, Lauren G. Zacharias, Andrew S. Chung, Qian Yang, Milind D. Chalishazar, Abbie S. Ireland, C. Allison Stewart, Kasey R. Cargill, Luc Girard, Yi Liu, Min Ni, Jian Xu, Xudong Wu, Hao Zhu, Benjamin J. Drapkin, Lauren A. Byers, Trudy G. Oliver, Adi Gazdar, John Minna, Ralph DeBerardinis

Lung megakaryocytes are immune modulatory cells

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Abstract

While platelets are the cellular mediators of thrombosis, platelets are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered as bone marrow (BM) resident cells. However, platelet producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared to BM Mks. We therefore sought to define the immune functions of lung Mks. Using single cell RNA-Seq of BM and lung myeloid enriched cells, we found that lung Mks (MkL) had gene expression patterns that are similar to antigen presenting cells (APC). This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment as evidenced by BM Mks having a MkL like phenotype under the influence of pathogen receptor challenge and lung associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that MkL internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, MkL induced CD4+ T cell activation in a MHC II dependent manner both in vitro and in vivo. These data indicated that Mks in the lung had key immune regulatory roles dictated in part by the tissue environment.

Authors

Daphne N. Pariser, Zachary T. Hilt, Sara K. Ture, Sara K. Blick-Nitko, Mark R. Looney, Simon J. Cleary, Estheany Roman-Pagan, Jerry Saunders II, Steve N. Georas, Janelle M. Veazey, Ferralita Madere, Laura Tesoro Santos, Allison M. Arne, Nguyen PT Huynh, Alison C. Livada, Selena M. Guerrero-Martin, Claire E. Lyons, Kelly A. Metcalf Pate, Kathleen E. McGrath, James Palis, Craig Morrell

Galectin‐7 downregulation in lesional keratinocytes contributes to enhanced IL‐17A signaling and skin pathology in psoriasis

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Abstract

Psoriasis is a chronic inflammatory skin disease characterized by inflammatory cell infiltration, as well as hyperproliferation of keratinocytes in skin lesions, and is considered a metabolic syndrome. We found that the expression of galectin-7 is reduced in the skin lesions of patients with psoriasis. IL-17A and TNF-α, two cytokines intimately involved in the development of psoriatic lesions, suppressed galectin-7 expression in human primary keratinocytes (HEKn cells) and the immortalized human keratinocyte cell line HaCaT. A galectin-7 knockdown in these cells elevated the production of IL-6 and IL-8 and enhanced ERK signaling when the cells were stimulated with IL-17A. Galectin-7 attenuated IL-17A–induced production of inflammatory mediators by keratinocytes via the miR-146a–ERK pathway. Moreover, galectin-7–deficient mice showed enhanced epidermal hyperplasia and skin inflammation in response to intradermal IL-23 injection. We identified fluvastatin as an inducer of galectin-7 expression by connectivity map (cMAP) analysis, confirmed this effect in keratinocytes, and demonstrated that fluvastatin attenuated IL-6 and IL-8 production induced by IL-17A. Thus, we validate a role of galectin-7 in the pathogenesis of psoriasis, in both epidermal hyperplasia and keratinocyte-mediated inflammatory responses, and formulated a rationale for the use of statins in the treatment of psoriasis.

Authors

Hung-Lin Chen, Chia-Hui Lo, Chi-Chun Huang, Meng-Ping Lu, Po-Yuan Hu, Chang-Shan Chen, Di-Yen Chueh, Peilin Chen, Teng-Nan Lin, Yuan-Hsin Lo, Yu-Ping Hsiao, Daniel K. Hsu, Fu-Tong Liu

Identification of human long non-coding RNAs associated with nonalcoholic fatty liver disease and metabolic homeostasis

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Abstract

A growing number of long non-coding RNAs (lncRNAs) have emerged as vital metabolic regulators. However, most human lncRNAs are non-conserved and highly tissue-specific, vastly limiting our ability to identify human lncRNA metabolic regulators (hLMRs). In this study, we establish a pipeline to identify putative hLMRs that are metabolically sensitive, disease-relevant, and population applicable. We first progressively processed multilevel human transcriptome data to select liver lncRNAs that exhibit highly dynamic expression in the general population, show differential expression in a nonalcoholic fatty liver disease (NAFLD) population, and response to dietary intervention in a small NAFLD cohort. We then experimentally demonstrated the responsiveness of selected hepatic lncRNAs to defined metabolic milieus in a liver-specific humanized mouse model. Furthermore, by extracting a concise list of protein-coding genes that are persistently correlated with lncRNAs in general and NAFLD populations, we predicted the specific function for each hLMR. Using gain- and loss-of-function approaches in humanized mice as well as ectopic expression in conventional mice, we validated the regulatory role of one non-conserved hLMR in cholesterol metabolism by coordinating with an RNA-binding protein, PTBP1, to modulate the transcription of cholesterol synthesis genes. Our work overcome the heterogeneity intrinsic to human data to enable the efficient identification and functional definition of disease-relevant human lncRNAs in metabolic homeostasis.

Authors

Xiangbo Ruan, Ping Li, Yonghe Ma, Chengfei Jiang, Yi Chen, Yu Shi, Nikhil Gupta, Fayaz Seifuddin, Mehdi Pirooznia, Yasuyuki Ohnishi, Nao Yoneda, Megumi Nishiwaki, Gabrijela Dumbovic, John L. Rinn, Yuichiro Higuchi, Kenji Kawai, Hiroshi Suemizu, Haiming Cao

Mutant SF3B1 promotes AKT and NF-kB driven mammary tumorigenesis

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Abstract

Mutations in the core RNA splicing factor SF3B1 are prevalent in leukemias and uveal melanoma but hotspot SF3B1 mutations are also seen in epithelial malignancies such as breast cancer. Although hotspot mutations in SF3B1 alter hematopoietic differentiation, whether SF3B1 mutations contribute to epithelial cancer development and progression is unknown. Here, we identify that SF3B1 mutations in mammary epithelial and breast cancer cells induce a recurrent pattern of aberrant splicing leading to activation of AKT and NF-kB, enhanced cell migration, and accelerated tumorigenesis. Transcriptomic analysis of human cancer specimens, MMTV-cre Sf3b1K700E/WT mice, and isogenic mutant cell lines identified hundreds of aberrant 3’ splice sites (3’ss) induced by mutant SF3B1. Consistently between mouse and human tumors, mutant SF3B1 promoted aberrant splicing (dependent on aberrant branchpoints as well as pyrimidines downstream of the cryptic 3’ss) and consequent suppression of PPP2R5A and MAP3K7, critical negative regulators of AKT and NF-kB. Coordinate activation of NF-kB and AKT signaling was observed in the knock-in models, leading to accelerated cell migration and tumor development in combination with mutant PIK3CA but also hypersensitizing cells to AKT kinase inhibitors. These data identify hotspot mutations in SF3B1 as an important contributor to breast tumorigenesis and reveal unique vulnerabilities in cancers harboring them.

Authors

Bo Liu, Zhaoqi Liu, Sisi Chen, Michelle Ki, Caroline Erickson, Jorge S. Reis-Filho, Benjamin H. Durham, Qing Chang, Elisa de Stanchina, Yiwei Sun, Raul Rabadan, Omar Abdel-Wahab, Sarat Chandarlapaty

Hypothalamic REV-ERB nuclear receptors control diurnal food intake and leptin sensitivity in diet-induced obese mice

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Abstract

Obesity occurs when energy expenditure is outweighed by energy intake. Tuberal hypothalamic nuclei, including the arcuate nucleus (ARC), ventromedial nucleus (VMH), and dorsomedial nucleus (DMH), control for food intake and energy expenditure. Here we reported that, contrary to females, male mice lacking circadian nuclear receptors REV-ERB alpha and beta in the tuberal hypothalamus (HDKO) gained excessive weight on an obesogenic high fat diet due to both decreased energy expenditure and increased food intake during the light phase. Moreover, rebound food intake after fasting was markedly increased in HDKO mice. Integrative transcriptomic and cistromic analyses revealed that such disruption in feeding behavior was due to perturbed REV-ERB-dependent leptin signaling in the ARC. Indeed, in vivo leptin sensitivity was impaired in HDKO mice on an obesogenic diet in a diurnal manner. Thus, REV-ERBs play a crucial role in hypothalamic control of food intake and diurnal leptin sensitivity in diet-induced obesity.

Authors

Marine Adlanmerini, Hoang C. B. Nguyen, Brianna M. Krusen, Clare W. Teng, Caroline E. Geisler, Lindsey C. Peed, Bryce J. Carpenter, Matthew R. Hayes, Mitchell A. Lazar