Characterization of the cell cycle–regulated transcripts in U2OS cells yielded 1871 unique genes. FOXM1 targets were identified via ChIP-seq, and novel targets in G2/M and S phases were verified using a real-time luciferase assay. ChIP-seq data were used to map cell cycle transcriptional regulators of cell cycle–regulated gene expression in U2OS cells.

Heterogeneity in systemic sclerosis/SSc confounds clinical trials. We previously identified ‘intrinsic’ gene expression subsets by analysis of SSc skin. Here we test the hypotheses that skin gene expression signatures including intrinsic subset are associated with skin score/MRSS improvement during mycophenolate mofetil (MMF) treatment. Gene expression and intrinsic subset assignment were measured in 12 SSc patients’ biopsies and ten controls at baseline, and from serial biopsies of one cyclophosphamide-treated patient, and nine MMF-treated patients. Gene expression changes during treatment were determined using paired t-tests corrected for multiple hypothesis testing. MRSS improved in four of seven MMF-treated patients classified as the inflammatory intrinsic subset. Three patients without MRSS improvement were classified as normal-like or fibroproliferative intrinsic subsets. 321 genes (FDR <5%) were differentially expressed at baseline between patients with and without MRSS improvement during treatment. Expression of 571 genes (FDR <10%) changed between pre- and post-MMF treatment biopsies for patients demonstrating MRSS improvement. Gene expression changes in skin are only seen in patients with MRSS improvement. Baseline gene expression in skin, including intrinsic subset assignment, may identify SSc patients whose MRSS will improve during MMF treatment, suggesting that gene expression in skin may allow targeted treatment in SSc.

Systemic sclerosis (SSc) is a rare systemic autoimmune disease characterized by skin and organ fibrosis. The pathogenesis of SSc and its progression are poorly understood. The SSc intrinsic gene expression subsets (inflammatory, fibroproliferative, normal-like, and limited) are observed in multiple clinical cohorts of patients with SSc. Analysis of longitudinal skin biopsies suggests that a patient's subset assignment is stable over 6–12 months. Genetically, SSc is multi-factorial with many genetic risk loci for SSc generally and for specific clinical manifestations. Here we identify the genes consistently associated with the intrinsic subsets across three independent cohorts, show the relationship between these genes using a gene-gene interaction network, and place the genetic risk loci in the context of the intrinsic subsets. To identify gene expression modules common to three independent datasets from three different clinical centers, we developed a consensus clustering procedure based on mutual information of partitions, an information theory concept, and performed a meta-analysis of these genome-wide gene expression datasets. We created a gene-gene interaction network of the conserved molecular features across the intrinsic subsets and analyzed their connections with SSc-associated genetic polymorphisms. The network is composed of distinct, but interconnected, components related to interferon activation, M2 macrophages, adaptive immunity, extracellular matrix remodeling, and cell proliferation. The network shows extensive connections between the inflammatory- and fibroproliferative-specific genes. The network also shows connections between these subset-specific genes and 30 SSc-associated polymorphic genes including STAT4, BLK, IRF7, NOTCH4, PLAUR, CSK, IRAK1, and several human leukocyte antigen (HLA) genes. Our analyses suggest that the gene expression changes underlying the SSc subsets may be long-lived, but mechanistically interconnected and related to a patients underlying genetic risk.

IntroductionSystemic sclerosis is an autoimmune disease characterized by inflammation and fibrosis of the skin and internal organs. We sought to assess the clinical and molecular effects associated with response to intravenous abatacept in patients with diffuse cutaneous systemic.MethodsAdult diffuse cutaneous systemic sclerosis patients were randomized in a 2:1 double-blinded fashion to receive abatacept or placebo over 24 weeks. Primary outcomes were safety and the change in modified Rodnan Skin Score (mRSS) at week 24 compared with baseline. Improvers were defined as patients with a decrease in mRSS of ≥30 % post-treatment compared to baseline. Skin biopsies were obtained for differential gene expression and pathway enrichment analyses and intrinsic gene expression subset assignment.ResultsTen subjects were randomized to abatacept (n = 7) or placebo (n = 3). Disease duration from first non-Raynaud’s symptom was significantly longer (8.8 ± 3.8 years vs. 2.4 ± 1.6 years, p = 0.004) and median mRSS was higher (30 vs. 22, p = 0.05) in the placebo compared to abatacept group. Adverse events were similar in the two groups. Five out of seven patients (71 %) randomized to abatacept and one out of three patients (33 %) randomized to placebo experienced ≥30 % improvement in skin score. Subjects receiving abatacept showed a trend toward improvement in mRSS at week 24 (−8.6 ± 7.5, p = 0.0625) while those in the placebo group did not (−2.3 ± 15, p = 0.75). After adjusting for disease duration, mRSS significantly improved in the abatacept compared with the placebo group (abatacept vs. placebo mRSS decrease estimate −9.8, 95 % confidence interval −16.7 to −3.0, p = 0.0114). In the abatacept group, the patients in the inflammatory intrinsic subset showed a trend toward greater improvement in skin score at 24 weeks compared with the patients in the normal-like intrinsic subset (−13.5 ± 3.1 vs. −4.5 ± 6.4, p = 0.067). Abatacept resulted in decreased CD28 co-stimulatory gene expression in improvers consistent with its mechanism of action. Improvers mapped to the inflammatory intrinsic subset and showed decreased gene expression in inflammatory pathways, while non-improver and placebos showed stable or reverse gene expression over 24 weeks.ConclusionsClinical improvement following abatacept therapy was associated with modulation of inflammatory pathways in skin.Trial registrationClinicalTrials.gov NCT00442611. Registered 1 March 2007.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0669-3) contains supplementary material, which is available to authorized users.

BackgroundSystemic sclerosis (SSc) is a multi-organ autoimmune disease characterized by skin fibrosis. Internal organ involvement is heterogeneous. It is unknown whether disease mechanisms are common across all involved affected tissues or if each manifestation has a distinct underlying pathology.MethodsWe used consensus clustering to compare gene expression profiles of biopsies from four SSc-affected tissues (skin, lung, esophagus, and peripheral blood) from patients with SSc, and the related conditions pulmonary fibrosis (PF) and pulmonary arterial hypertension, and derived a consensus disease-associate signature across all tissues. We used this signature to query tissue-specific functional genomic networks. We performed novel network analyses to contrast the skin and lung microenvironments and to assess the functional role of the inflammatory and fibrotic genes in each organ. Lastly, we tested the expression of macrophage activation state-associated gene sets for enrichment in skin and lung using a Wilcoxon rank sum test.ResultsWe identified a common pathogenic gene expression signature—an immune–fibrotic axis—indicative of pro-fibrotic macrophages (MØs) in multiple tissues (skin, lung, esophagus, and peripheral blood mononuclear cells) affected by SSc. While the co-expression of these genes is common to all tissues, the functional consequences of this upregulation differ by organ. We used this disease-associated signature to query tissue-specific functional genomic networks to identify common and tissue-specific pathologies of SSc and related conditions. In contrast to skin, in the lung-specific functional network we identify a distinct lung-resident MØ signature associated with lipid stimulation and alternative activation. In keeping with our network results, we find distinct MØ alternative activation transcriptional programs in SSc-associated PF lung and in the skin of patients with an “inflammatory” SSc gene expression signature.ConclusionsOur results suggest that the innate immune system is central to SSc disease processes but that subtle distinctions exist between tissues. Our approach provides a framework for examining molecular signatures of disease in fibrosis and autoimmune diseases and for leveraging publicly available data to understand common and tissue-specific disease processes in complex human diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-017-0417-1) contains supplementary material, which is available to authorized users.

Acquired resistance to BRAFV600E inhibitors (BRAFi) in melanoma remains a common clinical obstacle, as is the case for any targeted drug therapy that can be developed given the plastic nature of cancers. While there has been significant focus on the cancer cell-intrinsic properties of BRAFi resistance, the impact of BRAFi resistance on host immunity has not been explored. Here we provide preclinical evidence that resistance to BRAFi in an autochthonous mouse model of melanoma is associated with restoration of myeloid-derived suppressor cells (MDSC) in the tumor microenvironment initially reduced by BRAFi treatment. In contrast to restoration of MDSC, levels of T regulatory cells remained reduced in BRAFi-resistant tumors. Accordingly, tumor gene expression signatures specific for myeloid cell chemotaxis and homeostasis reappeared in BRAFi-resistant tumors. Notably, MDSC restoration relied upon MAPK pathway reactivation and downstream production of the myeloid attractant CCL2 in BRAFi-resistant melanoma cells. Strikingly, while combination checkpoint blockade (anti-CTLA-4 + anti-PD-1) was ineffective against BRAFi-resistant melanomas, the addition of MDSC depletion/blockade (anti-Gr-1 + CCR2 antagonist) prevented outgrowth of BRAFi-resistant tumors. Our results illustrate how extrinsic pathways of immunosuppression elaborated by melanoma cells dominate the tumor microenvironment and highlight the need to target extrinsic as well as intrinsic mechanisms of drug resistance.

IntroductionTyrosine kinase inhibitors (TKI) are medications of interest in the treatment of Systemic Sclerosis (SSc) because of their ability to inhibit pathways involved in fibrosis. In this open-label pilot trial, our objectives were to assess the safety, efficacy, and molecular change associated with treatment of patients with diffuse cutaneous (dc)SSc with the TKI nilotinib (Tasigna™).MethodsTen adult patients with early dcSSc were treated with nilotinib. Primary endpoints were safety and change in modified Rodnan Skin Score (MRSS) after 6 months. Lesional skin biopsies at baseline, 6 and 12 months of treatment were assessed by histopathology, immunohistochemistry, and DNA microarray.ResultsPatients had early and active dcSSc with median disease duration of 0.7 years (range 0.5, 1.7) and increasing MRSS in the month prior to baseline (mean +2.9, p=0.02). Seven out of ten patients completed 6 and 12 months of treatment. Seventy-one adverse events (AEs) including 2 serious AEs were observed, and 92 % of AEs were grade 1-2. Two patients discontinued the medication due to mild QTc prolongation. MRSS improved by a mean of 4.2 points (16 %) at 6 months and by 6.3 points (23 %) at 12 months in the 7 completers, p=0.02 and 0.01, respectively. Patients with a decrease in MRSS >20 % from baseline at 12 months (classified as improvers) had significantly higher expression of transforming growth factor beta receptor (TGFBR) and platelet-derived growth factor receptor beta (PDGFRB) signaling genes at baseline than non-improvers, and the expression of these genes significantly decreased in improvers post-treatment.ConclusionNilotinib was well tolerated by the majority of patients in this study, with tolerability limited primarily by mild QTc-prolongation. Significant MRSS improvement was observed in these early, active patients, but is not conclusive of treatment effect given the open-label study-design and small number of patients in this pilot study. Improvers had higher levels of expression of genes associated with TGFBR and PDGFRB signaling at baseline, and a significant decrease in the expression of these genes occurred only in patients with higher MRSS improvement. The findings of this pilot study warrant more conclusive evaluation.Trial registrationClinicaltrials.gov NCT01166139, July 1, 2010.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0721-3) contains supplementary material, which is available to authorized users.

Objective To assess the safety and efficacy of lenabasum in diffuse cutaneous systemic sclerosis (dc SS c). Methods A randomized, double‐blind, placebo‐controlled, phase II study was conducted at 9 SS c clinics in the US . Adults with dc SS c of ≤6 years’ duration who were receiving stable standard‐of‐care treatment were randomized to receive lenabasum (n = 27) or placebo (n = 15). Lenabasum doses were 5 mg once daily, 20 mg once daily, or 20 mg twice daily for 4 weeks, followed by 20 mg twice daily for 8 weeks. Safety and efficacy were assessed at weeks 4, 8, 12, and 16. Results Adverse events ( AE s) occurred in 63% of the lenabasum group and 60% of the placebo group, with no serious AE s related to lenabasum. Compared to placebo, lenabasum treatment was associated with greater improvement in the American College of Rheumatology Combined Response Index in diffuse cutaneous Systemic Sclerosis ( CRISS ) score and other efficacy outcome measures that assessed overall disease, skin involvement, and patient‐reported function. The median CRISS score increased in the lenabasum group during the study, reaching 0.33, versus 0.00 in the placebo group, at week 16 ( P = 0.07 by 2‐sided mixed‐effects model repeated‐measures analysis). Gene expression in inflammation and fibrosis pathways was reduced, and inflammation and fibrosis were improved on histologic evaluation of skin biopsy specimens, in the lenabasum group compared to the placebo group (all P ≤ 0.05). Conclusion Despite a short trial duration in a small number of patients in this phase II study in dc SS c, our findings indicate that lenabasum improves efficacy outcomes and underlying disease pathology with a favorable safety profile.