Virtually all patients with BRAF-mutant melanoma develop resistance to MAPK inhibitors largely through non-mutational events. Although the epigenetic landscape is shown to be altered in therapy-resistant melanomas and other cancers, a specific targetable epigenetic mechanism has not been validated to date. Here, we evaluate the CoREST repressor complex and the recently developed bivalent inhibitor, corin, within the context of melanoma phenotype plasticity and therapeutic resistance. We find that CoREST is a critical mediator of the major distinct melanoma phenotypes and that corin treatment of melanoma cells leads to phenotype reprogramming. Global assessment of transcript and chromatin changes conferred by corin reveals specific effects on histone marks connected to EMT-associated transcription factors and the dual-specificity phosphatases (DUSPs). Remarkably, treatment of BRAF inhibitor (BRAFi)-resistant melanomas with corin promotes resensitization to BRAFi therapy. DUSP1 is consistently downregulated in BRAFi-resistant melanomas which is reversed by corin treatment and associated with inhibition of p38 MAPK activity and resensitization to BRAFi therapies. Moreover, this activity can be recapitulated by the p38 MAPK inhibitor, BIRB 796. These findings identify the CoREST repressor complex as a central mediator of melanoma phenotype plasticity and resistance to targeted therapy and suggest that CoREST inhibitors may prove beneficial to patients with BRAFi-resistant melanoma.
Muzhou Wu, Ailish Hanly, Frederick Gibson, Robert Fisher, Samantha Rogers, Kihyun Park, Angelina Zuger, Kevin Kuang, Jay H. Kalin, Sarah Nocco, Matthew Cole, Amy Xiao, Filisia Agus, Adam Labadorf, Samuel Beck, Marianne Collard, Philip A. Cole, Rhoda M. Alani
Patients with chronic inflammatory disorders such as psoriasis have an increased risk of cardiovascular disease and elevated levels of LL37, a cathelicidin host defense peptide that has both antimicrobial and proinflammatory properties. To explore if LL37 could contribute to the risk of heart disease, we examined its effects on lipoprotein metabolism and show that LL37 enhances LDL uptake in macrophages through LDLR, SR-B1 and CD36. This interaction led to increased cytosolic cholesterol in macrophages and changes in expression of lipid metabolism genes consistent with increased cholesterol uptake. Structure-function analysis and synchrotron small angle X-ray scattering show structural determinants of the LL37-LDL complex that underlie its ability to bind its receptors and promote uptake. This function of LDL uptake is unique to cathelicidins from humans and some primates and was not observed with cathelicidins from mice or rabbits. Notably, Apoe-/- mice expressing LL37 develop larger atheroma plaques than control mice and a positive correlation between plasma LL37 and OxPL-apoB levels was observed in human subjects with cardiovascular disease. These findings provide evidence that LDL uptake can be increased via interaction with LL37 and may explain the increased risk of cardiovascular disease associated with the chronic inflammatory disorders.
Yoshiyuki Nakamura, Nikhil N. Kulkarni, Toshiya Takahashi, Haleh Alimohamadi, Tatsuya Dokoshi, Edward L. Liu, Michael Shia, Tomofumi Numata, Elizabeth W.C. Luo, Adrian F. Gombart, Xiaohong Yang, Patrick Secrest, Philip L.S.M. Gordts, Sotirios Tsimikas, Gerard C.L. Wong, Richard L. Gallo
Aplasia cutis congenita (ACC) is a congenital epidermal defect of the midline scalp and has been proposed to be due to a primary keratinocyte abnormality. Why it forms mainly at this anatomic site has remained a longstanding enigma. KCTD1 mutations cause ACC, ectodermal abnormalities, and kidney fibrosis, whereas KCTD15 mutations cause ACC and cardiac outflow tract abnormalities. Here, we find that KCTD1 and KCTD15 can form multimeric complexes and can compensate for each other's loss, and that disease mutations are dominant-negative, resulting in lack of KCTD1/KCTD15 function. We demonstrate that KCTD15 is critical for cardiac outflow tract development, whereas KCTD1 regulates distal nephron function. Combined inactivation of KCTD1/KCTD15 in keratinocytes results in abnormal skin appendages, but not in ACC. Instead, KCTD1/KCTD15 inactivation in neural crest cells results in ACC linked to midline skull defects, demonstrating that ACC is not caused by a primary defect in keratinocytes but is a secondary consequence of impaired cranial neural crest cells giving rise to midline cranial suture cells that express keratinocyte-promoting growth factors. Our findings explain the clinical observations in patients with KCTD1 versus KCTD15 mutations, establish KCTD1/KCTD15 as critical regulators of ectodermal and neural crest cell functions, and define ACC as a neurocristopathy.
Jackelyn R. Raymundo, Hui Zhang, Giovanni Smaldone, Wenjuan Zhu, Kathleen E. Daly, Benjamin J. Glennon, Giovanni Pecoraro, Marco Salvatore, William A. Devine, Cecilia W. Lo, Luigi Vitagliano, Alexander G. Marneros
Hidradenitis suppurativa (HS) is a chronic inflammatory disease characterized by abscesses, nodules, dissecting/draining tunnels, and extensive fibrosis. Here, we integrate single-cell RNA sequencing, spatial transcriptomics, and immunostaining to provide an unprecedented view of the pathogenesis of chronic HS, characterizing the main cellular players, and defining their interactions. We describe a striking layering of the chronic HS infiltrate and identify the contribution of two fibroblast subtypes (SFRP4+ and CXCL13+) in orchestrating this compartmentalized immune response. We further demonstrate the central role of the Hippo pathway in promoting extensive fibrosis in HS and provide pre-clinical evidence that the pro-fibrotic fibroblast response in HS can be modulated through inhibition of this pathway. These data provide novel insights into key aspects of HS pathogenesis with broad therapeutic implications.
Kelsey R. van Straalen, Feiyang Ma, Pei-Suen Tsou, Olesya Plazyo, Mehrnaz Gharaee-Kermani, Marta Calbet, Xianying Xing, Mrinal K. Sarkar, Ranjitha Uppala, Paul W. Harms, Rachael Wasikowski, Lina Nahlawi, Mio Nakamura, Milad Eshaq, Cong Wang, Craig J. Dobry, Jeffrey H. Kozlow, Jill R. Cherry-Bukowiec, William D. Brodie, Kerstin Wolk, Özge Uluckan, Megan N. Mattichak, Matteo Pellegrini, Robert L. Modlin, Emanual Maverakis, Robert Sabat, J. Michelle Kahlenberg, Allison C. Billi, Lam C. Tsoi, Johann E. Gudjonsson
Sarcoidosis is a disease of unknown etiology in which granulomas form throughout the body and is typically treated with glucocorticoids, but there are no approved steroid-sparing alternatives. Here, we investigated the mechanism of granuloma formation using single-cell RNA-Seq in sarcoidosis patients. We observed that the percentages of triggering receptor expressed on myeloid cells 2–positive (TREM2-positive) macrophages expressing angiotensin-converting enzyme (ACE) and lysozyme, diagnostic makers of sarcoidosis, were increased in cutaneous sarcoidosis granulomas. Macrophages in the sarcoidosis lesion were hypermetabolic, especially in the pentose phosphate pathway (PPP). Expression of the PPP enzymes, such as fructose-1,6-bisphosphatase 1 (FBP1), was elevated in both systemic granuloma lesions and serum of sarcoidosis patients. Granuloma formation was attenuated by the PPP inhibitors in in vitro giant cell and in vivo murine granuloma models. These results suggest that the PPP may be a promising target for developing therapeutics for sarcoidosis.
Satoshi Nakamizo, Yuki Sugiura, Yoshihiro Ishida, Yoko Ueki, Satoru Yonekura, Hideaki Tanizaki, Hiroshi Date, Akihiko Yoshizawa, Teruasa Murata, Kenji Minatoya, Mikako Katagiri, Seitaro Nomura, Issei Komuro, Seishi Ogawa, Saeko Nakajima, Naotomo Kambe, Gyohei Egawa, Kenji Kabashima
Background. Pemphigus, a rare autoimmune bullous disease mediated by anti-desmoglein autoantibodies, can be controlled with systemic medication like rituximab and high-dose systemic corticosteroids combined with immunosuppressants. However, some patients continue to experience chronically recurrent blisters which require long-term maintenance systemic therapy. METHODS. Skin with chronic blisters was obtained from patients with pemphigus. Immunologic properties of the skin were analyzed by immunofluorescence staining, bulk and single-cell RNA and TCR sequencing, and a highly multiplex imaging technique known as CO-Detection by indEXing (CODEX). Functional analyses were performed by flow cytometry and bulk RNA-sequencing using peripheral blood from healthy donors. Intralesional corticosteroid was injected into patient skin, and changes in chronically recurrent blisters were observed. RESULTS. We demonstrate the presence of skin tertiary lymphoid structures (TLSs) with desmoglein-specific B cells in chronic blisters from pemphigus patients. In the skin TLSs, CD4+ T cells predominantly produced CXCL13. These clonally expanded CXCL13+CD4+ T cells exhibited features of activated Th1-like cells and downregulated genes associated with T-cell receptor-mediated signaling. Regulatory T cells (Tregs) are in direct contact with CXCL13+CD4+ memory T cells and increased CXCL13 production of CD4+ T cells through IL-2 consumption and TGF-β stimulation. Lastly, Intralesional corticosteroid injection improved chronic blisters and reduce skin TLSs in patients with pemphigus. CONCLUSIONS. This study concludes that skin TLSs are associated with the persistence of chronically recurrent blisters in pemphigus patients, and the microenvironmental network involving CXCL13+CD4+ T cells and Tregs within these structures plays an important role in CXCL13 production. TRIAL REGISTRATION. NCT04509570 FUNDING. This work was supported by National Research Foundation of South Korea (grant NRF-2021R1C1C1007179) and Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (grant RS-2022-00165917).
Dawoon Han, A. Yeong Lee, Taehee Kim, Ji Young Choi, Mi Yeon Cho, Ahreum Song, Changhyeon Kim, Joon Ho Shim, Hyun Je Kim, Honesty Kim, Hillary Blaize D'Angio, Ryan Preska, Aaron T. Mayer, Miri Kim, Eun-Ji Choi, Tae-Gyun Kim, Eui-Cheol Shin, Kyemyung Park, Do-Young Kim, Soo-Chan Kim, Jong Hoon Kim
The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in macrophages, but whether they have distinct functions remains unclear. We confirmed that GLUT1 expression increased after M1 polarization stimuli and found that GLUT3 expression increased after M2 stimulation in macrophages. Conditional deletion of Glut3 (LysM-Cre Glut3fl/fl) impaired M2 polarization of bone marrow derived macrophages. Alternatively activated macrophages from the skin of atopic dermatitis patients showed increased GLUT3 expression, and a calcipotriol-induced model of atopic dermatitis was rescued LysM-Cre Glut3fl/fl mice. M2-like macrophages expressed GLUT3 in human wound tissues as assessed by transcriptomics and co-staining, and GLUT3 expression was significantly decreased in non-healing, compared with healing, diabetic foot ulcers. In an excisional wound healing model, LysM-Cre Glut3fl/fl mice showed significantly impaired M2 macrophage polarization and delayed wound healing. GLUT3 promoted IL-4/STAT6 signaling, independent from its glucose transport activity. Unlike plasma membrane-localized GLUT1, GLUT3 was localized primarily to endosomes and was required for the efficient endocytosis of IL4Ra subunits. GLUT3 interacted directly with GTP-bound RAS in vitro and in vivo through its intracytoplasmic loop domain (ICH), and this interaction was required for efficient STAT6 activation and M2 polarization. PAK activation and macropinocytosis were also impaired without GLUT3, suggesting broader roles for GLUT3 in the regulation of endocytosis. Thus, GLUT3 is required for efficient alternative macrophage polarization and function, through a glucose transport-independent, RAS-mediated role in the regulation of endocytosis and IL-4/STAT6 activation.
Dong-Min Yu, Jiawei Zhao, Eunice E. Lee, Dohun Kim, Ruchika Mahapatra, Elysha K. Rose, Zhiwei Zhou, Calvin R. Hosler, Abdullah El-Kurdi, Jun-yong Choe, E. Dale Abel, Gerta Hoxhaj, Kenneth D. Westover, Raymond J. Cho, Jeffrey B. Cheng, Richard C. Wang
CRISPR-Cas9 has been proposed as a treatment for genetically inherited skin disorders. Here we report that CRISPR transfection activates STING-dependent antiviral responses in keratinocytes, resulting in heightened endogenous interferon (IFN) responses through induction of IFN-κ leading to decreased plasmid stability secondary to induction of the cytidine deaminase APOBEC3G. Notably, CRISPR-generated KO keratinocytes had permanent suppression of IFN-κ and IFN-stimulated gene (ISG) expression, secondary to hypermethylation of the IFNK promoter region by the DNA methyltransferase DNMT3B. JAK inhibition via baricitinib prior to CRISPR transfection increased transfection efficiency, prevented IFNK promoter hypermethylation, and restored normal IFN-κ activity and ISG responses. This work shows that CRISPR-mediated gene correction alters antiviral responses in keratinocytes, has implications for future gene therapies of inherited skin diseases using CRISPR technology, and suggests pharmacologic JAK inhibition as a tool for facilitating and attenuating inadvertent selection effects in CRISPR-Cas9 therapeutic approaches.
Mrinal K. Sarkar, Ranjitha Uppala, Chang Zeng, Allison C. Billi, Lam C. Tsoi, Austin Kidder, Xianying Xing, Bethany E. Perez White, Shuai Shao, Olesya Plazyo, Sirisha Sirobhushanam, Enze Xing, Yanyun Jiang, Katherine A. Gallagher, John J. Voorhees, J. Michelle Kahlenberg, Johann E. Gudjonsson
BACKGROUND. Merkel cell carcinoma (MCC) is an aggressive neuroendocrine (NE) skin cancer caused by severe UV-induced mutations or expression of Merkel cell polyomavirus (MCPyV) large and small T antigens (LT and ST). Despite deep genetic differences between MCPyV-positive and -negative subtypes, current clinical diagnostic markers are indistinguishable between subtypes and the expression profile of MCC tumors is unexplored. METHODS. Here we leveraged bulk and single-cell RNA sequencing of patient-derived tumor biopsies and cell lines to explore the underlying transcriptional diversity of MCC. RESULTS. Strikingly, MCC samples could be separated into transcriptional subtypes that were independent of MCPyV status. Instead, we observed an inverse correlation between a NE gene signature and the Hippo pathway transcription factors Yes1-associated transcriptional regulator (YAP1) and WW domain containing transcriptional regulator (WWTR1). This inverse correlation was present at the transcript and protein levels in the tumor biopsies as well as in established and patient-derived cell lines. Mechanistically, expression of YAP1 or WWTR1 in a MCPyV-positive MCC cell line induced cell-cycle arrest at least in part through TEAD-dependent transcriptional repression of MCPyV LT. CONCLUSION. These findings describe previously unrecognized heterogeneity in NE gene expression within MCC and support the model that YAP1/WWTR1 silencing is essential for the development of MCPyV-positive MCC. FUNDING. US Public Health Service grants R35CA232128, P01CA203655, and P30CA06516.
Thomas C. Frost, Ashley K. Gartin, Mofei Liu, Jingwei Cheng, Harita Dharaneeswaran, Derin B. Keskin, Catherine J. Wu, Anita Giobbie-Hurder, Manisha Thakuria, James A. DeCaprio
BACKGROUND. Acute febrile neutrophilic dermatosis (Sweet syndrome) is a potentially fatal multiorgan inflammatory disease characterized by fever, leukocytosis, and a rash with a neutrophilic infiltrate. Disease pathophysiology remains elusive, and current dogma suggests Sweet syndrome is a “reactive” process to an unknown antigen. Corticosteroids and steroid-sparing agents remain front-line therapies, but refractory cases pose a clinical challenge. METHODS. A 51-year-old woman with multiorgan Sweet syndrome developed serious corticosteroid-related side effects and was refractory to steroid-sparing agents. Blood counts, liver enzymes, and skin histopathology supported the diagnosis. Whole genome sequencing, transcriptomic profiling, and cellular assays of patient’s skin and neutrophils were performed. RESULTS. We identified elevated IL-1 signaling in lesional Sweet syndrome skin caused by a PIK3R1 gain-of-function mutation specifically found in neutrophils. This mutation increased neutrophil migration towards IL-1β and neutrophil respiratory burst. Targeted treatment with an IL-1R1 antagonist in the patient resulted in a dramatic therapeutic response and enabled tapering of corticosteroids. CONCLUSIONS. Dysregulated PI3K-AKT signaling is the first signaling pathway linked to Sweet syndrome and suggests Sweet syndrome may be caused by acquired mutations that modulate neutrophil function. Moreover, integration of molecular data across multiple levels identified a distinct subtype within a heterogenous disease that resulted in a rational and successful clinical intervention. Future cases will benefit from efforts to identify potential mutations. The ability to directly interrogate diseased skin allows this method to be generalizable to other inflammatory diseases and demonstrates a potential personalized medicine approach for challenging patients. FUNDING Berstein Foundation, NIH, VA, Moseley Foundation, and H.T. Leung Foundation.
Shreya Bhattacharya, Sayon Basu, Emily Sheng, Christina M. Murphy, Jenny Wei, Anna E. Kersh, Caroline A. Nelson, Joshua S. Bryer, Hovik A. Ashchyan, Katherine T. Steele, Amy K. Forrestel, John T. Seykora, Robert G. Micheletti, William D. James, Misha Rosenbach, Thomas H. Leung