J.C. Chen

Microenvironmental reprogramming by three-dimensional culture enables dermal papilla cells to induce de novo human hair-follicle growth

Significance Growth of de novo hair follicles in adult skin occurs by a process known as hair neogenesis. One way of initiating neogenesis is to place dermal papillae isolated from the hair follicle in contact with an overlying epidermis where they reprogram the epidermis to adopt a follicular fate. This approach, however, has not been successful using cultured human dermal papilla cells in human skin because the cells lose their ability to induce hair growth after expansion in vitro. In this paper, we demonstrate that by manipulating cell culture conditions to establish three-dimensional papilla spheroids, we restore dermal papilla inductivity. We also use several systems biology approaches to gain a comprehensive understanding of the molecular mechanisms that underlie this regenerative process. De novo organ regeneration has been observed in several lower organisms, as well as rodents; however, demonstrating these regenerative properties in human cells and tissues has been challenging. In the hair follicle, rodent hair follicle-derived dermal cells can interact with local epithelia and induce de novo hair follicles in a variety of hairless recipient skin sites. However, multiple attempts to recapitulate this process in humans using human dermal papilla cells in human skin have failed, suggesting that human dermal papilla cells lose key inductive properties upon culture. Here, we performed global gene expression analysis of human dermal papilla cells in culture and discovered very rapid and profound molecular signature changes linking their transition from a 3D to a 2D environment with early loss of their hair-inducing capacity. We demonstrate that the intact dermal papilla transcriptional signature can be partially restored by growth of papilla cells in 3D spheroid cultures. This signature change translates to a partial restoration of inductive capability, and we show that human dermal papilla cells, when grown as spheroids, are capable of inducing de novo hair follicles in human skin.

Identification of Causal Genetic Drivers of Human Disease through Systems-Level Analysis of Regulatory Networks

Identification of driver mutations in human diseases is often limited by cohort size and availability of appropriate statistical models. We propose a framework for the systematic discovery of genetic alterations that are causal determinants of disease, by prioritizing genes upstream of functional disease drivers, within regulatory networks inferred de novo from experimental data. We tested this framework by identifying the genetic determinants of the mesenchymal subtype of glioblastoma. Our analysis uncovered KLHL9 deletions as upstream activators of two previously established master regulators of the subtype, C/EBPβ and C/EBPδ. Rescue of KLHL9 expression induced proteasomal degradation of C/EBP proteins, abrogated the mesenchymal signature, and reduced tumor viability in vitro and in vivo. Deletions of KLHL9 were confirmed in > 50% of mesenchymal cases in an independent cohort, thus representing the most frequent genetic determinant of the subtype. The method generalized to study other human diseases, including breast cancer and Alzheimers disease.

Safety and efficacy of the JAK inhibitor tofacitinib citrate in patients with alopecia areata

BACKGROUND Alopecia areata (AA) is an autoimmune disease characterized by hair loss mediated by CD8+ T cells. There are no reliably effective therapies for AA. Based on recent developments in the understanding of the pathomechanism of AA, JAK inhibitors appear to be a therapeutic option; however, their efficacy for the treatment of AA has not been systematically examined. METHODS This was a 2-center, open-label, single-arm trial using the pan-JAK inhibitor, tofacitinib citrate, for AA with >50% scalp hair loss, alopecia totalis (AT), and alopecia universalis (AU). Tofacitinib (5 mg) was given twice daily for 3 months. Endpoints included regrowth of scalp hair, as assessed by the severity of alopecia tool (SALT), duration of hair growth after completion of therapy, and disease transcriptome. RESULTS Of 66 subjects treated, 32% experienced 50% or greater improvement in SALT score. AA and ophiasis subtypes were more responsive than AT and AU subtypes. Shorter duration of disease and histological peribulbar inflammation on pretreatment scalp biopsies were associated with improvement in SALT score. Drug cessation resulted in disease relapse in 8.5 weeks. Adverse events were limited to grade I and II infections. An AA responsiveness to JAK/STAT inhibitors score was developed to segregate responders and nonresponders, and the previously developed AA disease activity index score tracked response to treatment. CONCLUSIONS At the dose and duration studied, tofacitinib is a safe and effective treatment for severe AA, though it does not result in a durable response. Transcriptome changes reveal unexpected molecular complexity within the disease. TRIAL REGISTRATION ClinicalTrials.gov NCT02197455 and NCT02312882. FUNDING This work was supported by the US Department of Veterans Affairs Office of Research and Development, National Institute of Arthritis and Musculoskeletal and Skin Diseases National Institutes of Health grant R01 AR47223 and U01 AR67173, the National Psoriasis Foundation, the Swedish Society of Medicine, the Fernström Foundation, the Locks of Love Foundation, the National Alopecia Areata Foundation, and the Ranjini and Ajay Poddar Resource Fund for Dermatologic Diseases Research.

Pharmacologic inhibition of JAK-STAT signaling promotes hair growth.

Topical treatment of mouse and human skin with small-molecule inhibitors of the JAK-STAT pathway results in hair growth. Several forms of hair loss in humans are characterized by the inability of hair follicles to enter the growth phase (anagen) of the hair cycle after being arrested in the resting phase (telogen). Current pharmacologic therapies have been largely unsuccessful in targeting pathways that can be selectively modulated to induce entry into anagen. We show that topical treatment of mouse and human skin with small-molecule inhibitors of the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway results in rapid onset of anagen and subsequent hair growth. We show that JAK inhibition regulates the activation of key hair follicle populations such as the hair germ and improves the inductivity of cultured human dermal papilla cells by controlling a molecular signature enriched in intact, fully inductive dermal papillae. Our findings open new avenues for exploration of JAK-STAT inhibition for promotion of hair growth and highlight the role of this pathway in regulating the activation of hair follicle stem cells.

Molecular signatures define alopecia areata subtypes and transcriptional biomarkers

Alopecia areata (AA) is an autoimmune disease typified by nonscarring hair loss with a variable clinical course. In this study, we conducted whole genome gene expression analysis of 96 human scalp skin biopsy specimens from AA or normal control subjects. Based on gene expression profiling, samples formed distinct clusters based on the presence or absence of disease as well as disease phenotype (patchy disease compared with alopecia totalis or universalis). Differential gene expression analysis allowed us to robustly demonstrate graded immune activity in samples of increasing phenotypic severity and generate a quantitative gene expression scoring system that classified samples based on interferon and cytotoxic T lymphocyte immune signatures critical for disease pathogenesis.

An Open-Label Pilot Study to Evaluate the Efficacy of Tofacitinib in Moderate to Severe Patch-Type Alopecia Areata, Totalis, and Universalis

Alopecia areata (AA) is a common autoimmune disease with a lifetime risk of ∼2%. In AA, the immune system targets the hair follicle, resulting in clinical hair loss. The prognosis of AA is unpredictable, and currently there is no definitive treatment. Our previous whole genome expression studies identified active immune circuits in AA lesions, including common γ-chain cytokine and IFN pathways. Because these pathways are mediated through JAK kinases, we prioritized clinical exploration of small molecule JAK inhibitors. In preclinical trials in mice, tofacitinib successfully prevented AA development and reversed established disease. In our tofacitinib trial in 12 patients with moderate to severe AA, 11 patients completed a full course of treatment with minimal adverse events. Following limited response to the initial dose (5 mg b.i.d.), the dose was escalated (10 mg b.i.d.) for nonresponding subjects. Eight of 12 patients demonstrated ≥50% hair regrowth, while three patients demonstrated <50% hair regrowth, as measured by Severity in Alopecia Tool scoring. One patient demonstrated no regrowth. Gene expression profiles and Alopecia Areata Disease Activity Index scores correlated with clinical response. Our open-label studies of ruxolitinib and tofacitinib have shown dramatic clinical responses in moderate to severe AA, providing strong rationale for larger clinical trials using JAK inhibitors in AA. ClinicalTrials.gov ID NCT02299297.

DIGGIT: a Bioconductor package to infer genetic variants driving cellular phenotypes

UNLABELLED Identification of driver mutations in human diseases is often limited by cohort size and availability of appropriate statistical models. We propose a method for the systematic discovery of genetic alterations that are causal determinants of disease, by prioritizing genes upstream of functional disease drivers, within regulatory networks inferred de novo from experimental data. Here we present the implementation of Driver-gene Inference by Genetical-Genomic Information Theory as an R-system package. AVAILABILITY AND IMPLEMENTATION The diggit package is freely available under the GPL-2 license from Bioconductor (http://www.bioconductor.org).

Abstract PR10: Oncogenic dysregulations in neuroblastoma are associated with distal large chromosomal aberrations

Neuroblastoma (NB) is a prenatal malignancy diagnosed in infants, arising from neural crest cells, with heterogeneous etiology and prognosis. High risk tumors harbor large chromosomal alterations that substantially impact the expression of approximately one quarter of the genome; this challenges the distinction between driver and passenger copy number mutations, hindering the discovery of new therapeutic targets. Our lab has established a paradigm known as the bottleneck hypothesis in which multiple disease driver genetic elements integrate their aberrant signal through regulatory bottleneck, typically formed by a few transcriptional regulators, responsible of maintaining aggressive phenotypes. A new algorithm developed in our lab DIGGIT (Driver-Gene Inference by Genetical-Genomic Information Theory) has been able to successfully identify the impact of deletions in KLHL9 on the transcriptional activity of C/EBPβ and C/EBPδ, established master regulators of mesenchymal subtype in glioblastoma(1). To gain understanding of the association between genetics and the molecular phenotype which drives NB disease we analyze genome wide expression and copy number profiles from primary tumors from two independent cohorts with clinical information available; TARGET (n=250) and SIOPEN (n2=278); first, we identify genomic regions that prevalently suffer gain/loss aberrations which genetic dosage is associated with patient survival using a Cox hazards model. The main covariates are chromosome 1p (P=2.3e-9), 3p (P = 1.1e-2), 6q (P=), 11q (P=9.7e-7), 17p(P=5.3e-4) and 17q(P=4.5e-3). Also, we observed a multiplicative affect of chromosome 17 imbalance between p and q arms (P=7.8e-6). All measured with independence of MYCN amplification (the main hallmark of NB aggressive tumors). We used the above-mentioned dosage of these regions as trait loci to perform trans-aQTL analysis using the algorithm DIGGIT. We also included p and q arms combined cox linear model of chromosomes 11 and 17 to study the imbalance effect. The LIM domain only 1 protein (LMO1) is a validated NB oncogene which expression is increased in 9% of patients due to duplication events of its chromosome 11p15 locus. These events only partially explain LMO1 de-regulation. Our findings show that deletions on the distal arm 11q are implicated in LMO1 increased activity (P=4.3e-8), this effect is significantly stronger than its own loci duplication acting in cis (P=7.07e-5). The linear combination of p and q arms strikes with a p-value P=7.8e-10 supporting the additive effect of chromosome 11 imbalance. In addition to this finding, which confirms the oncogenic role of LMO1 in this disease, our integrated analysis also identified a plethora of additional findings providing plausible hypotheses for genetic alterations that contribute to dysregulation of driver genes in NB. The approach presented here is especially well suited to study tumors characterized by genomic instabilities leading to large chromosomal rearrangements, despite a paucity of recurrent point mutations. 1. James C. Chen, et al. Identification of Causal Genetic Drivers of Human Disease through Systems-Level Analysis of Regulatory Networks. Cell, Volume 159, Issue 2, p402–414, 9 October 2014. Citation Format: Gonzalo Lopez, Mariano Alvarez, James Chen, Presha Rajbhandari, Kristina A. Cole, Edward F. Attiyeh, Sharon Diskin, Pieter Mestdagh, Jo Vandesompele, John M. Maris, Andrea Califano. Oncogenic dysregulations in neuroblastoma are associated with distal large chromosomal aberrations. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr PR10.