Discovery of a Series of Pyrazinone RORγ Antagonists and Identification of the Clinical Candidate BI 730357.

The interleukin (IL)-23/T helper (Th)17 axis plays a critical role in autoimmune diseases, and there is an increasing number of biologic therapies that target IL-23 and IL-17. The transcription factor retinoic acid receptor-related orphan nuclear receptor γt (RORγt) is important for the activation and differentiation of Th17 cells and thus is an attractive pharmacologic target for the treatment of Th17-mediated diseases. A novel series of pyrazinone RORγ antagonists was discovered through hybridization of two distinct screening hits and scaffold hopping. The series offers attractive potency and selectivity in combination with favorable druglike properties, such as metabolic stability and aqueous solubility. Lead optimization identified a clinical candidate, compound (S)-11 (BI 730357), for the treatment of autoimmune diseases.

RORγt inhibition selectively targets IL-17 producing iNKT and γδ-T cells enriched in Spondyloarthritis patients.

Dysregulated IL-23/IL-17 responses have been linked to psoriatic arthritis and other forms of spondyloarthritides (SpA). RORγt, the key Thelper17 (Th17) cell transcriptional regulator, is also expressed by subsets of innate-like T cells, including invariant natural killer T (iNKT) and γδ-T cells, but their contribution to SpA is still unclear. Here we describe the presence of particular RORγt+T-betloPLZF- iNKT and γδ-hi T cell subsets in healthy peripheral blood. RORγt+ iNKT and γδ-hi T cells show IL-23 mediated Th17-like immune responses and were clearly enriched within inflamed joints of SpA patients where they act as major IL-17 secretors. SpA derived iNKT and γδ-T cells showed unique and Th17-skewed phenotype and gene expression profiles. Strikingly, RORγt inhibition blocked γδ17 and iNKT17 cell function while selectively sparing IL-22+ subsets. Overall, our findings highlight a unique diversity of human RORγt+ T cells and underscore the potential of RORγt antagonism to modulate aberrant type 17 responses.

Detecting copy number status and uncovering subclonal markers in heterogeneous tumor biopsies.

BACKGROUND: Genomic aberrations can be used to determine cancer diagnosis and prognosis. Clinically relevant novel aberrations can be discovered using high-throughput assays such as Single Nucleotide Polymorphism (SNP) arrays and next-generation sequencing, which typically provide aggregate signals of many cells at once. However, heterogeneity of tumor subclones dramatically complicates the task of detecting aberrations. RESULTS: The aggregate signal of a population of subclones can be described as a linear system of equations. We employed a measure of allelic imbalance and total amount of DNA to characterize each locus by the copy number status (gain, loss or neither) of the strongest subclonal component. We designed simulated data to compare our measure to existing approaches and we analyzed SNP-arrays from 30 melanoma samples and transcriptome sequencing (RNA-Seq) from one melanoma sample.We showed that any system describing aggregate subclonal signals is underdetermined, leading to non-unique solutions for the exact copy number profile of subclones. For this reason, our illustrative measure was more robust than existing Hidden Markov Model (HMM) based tools in inferring the aberration status, as indicated by tests on simulated data. This higher robustness contributed in identifying numerous aberrations in several loci of melanoma samples. We validated the heterogeneity and aberration status within single biopsies by fluorescent in situ hybridization of four affected and transcriptionally up-regulated genes E2F8, ETV4, EZH2 and FAM84B in 11 melanoma cell lines. Heterogeneity was further demonstrated in the analysis of allelic imbalance changes along single exons from melanoma RNA-Seq. CONCLUSIONS: These studies demonstrate how subclonal heterogeneity, prevalent in tumor samples, is reflected in aggregate signals measured by high-throughput techniques. Our proposed approach yields high robustness in detecting copy number alterations using high-throughput technologies and has the potential to identify specific subclonal markers from next-generation sequencing data.

MicroRNA signatures differentiate melanoma subtypes.

Melanoma is an aggressive cancer that is highly resistance to therapies once metastasized. We studied microRNA (miRNA) expression in clinical melanoma subtypes and evaluated different miRNA signatures in the background of gain of function somatic and inherited mutations associated with melanoma. Total RNA from 42 patient derived primary melanoma cell lines and three independent normal primary melanocyte cell cultures was evaluated by miRNA array. MiRNA expression was then analyzed comparing subtypes and additional clinicopathologic criteria including somatic mutations. The prevalence and association of an inherited variant in a miRNA binding site in the 3'UTR of the KRAS oncogene, referred to as the KRAS-variant, was also evaluated. We show that seven miRNAs, miR-142-3p, miR-486, miR-214, miR-218, miR-362, miR-650 and miR-31, were significantly correlated with acral as compared to non-acral melanomas (p < 0.04). In addition, we discovered that the KRAS-variant was enriched in non-acral melanoma (25%), and that miR-137 under expression was significantly associated with melanomas with the KRAS-variant. Our findings indicate that miRNAs are differentially expressed in melanoma subtypes and that their misregulation can be impacted by inherited gene variants, supporting the hypothesis that miRNA misregulation reflects biological differences in melanoma.

Plasma markers for identifying patients with metastatic melanoma.

PURPOSE: With the rising incidence of melanoma, more patients are undergoing surveillance for disease recurrence. Our purpose was to study levels of proteins that might be secreted in the blood of patients with metastatic melanoma that can be used for monitoring these individuals. METHODS: Genome-wide gene expression data were used to identify abundantly expressed genes in melanoma cells that encode for proteins likely to be present in the blood of cancer patients, based on high expression levels in tumors. ELISA assays were employed to measure proteins in plasma of 216 individuals; 108 metastatic melanoma patients and 108 age- and gender-matched patients with resected stage I/II disease split into equal-sized training and test cohorts. RESULTS: Levels of seven markers, CEACAM (carcinoembryonic antigen-related cell adhesion molecule), ICAM-1 (intercellular adhesion molecule 1), osteopontin, MIA (melanoma inhibitory activity), GDF-15 (growth differentiation factor 15), TIMP-1 (tissue inhibitor of metalloproteinase 1), and S100B, were higher in patients with unresected stage IV disease than in patients with resected stage I/II disease. About 81% of the stage I/II patients in the training set had no marker elevation, whereas 69% of the stage IV patients had elevation of at least one marker (P < 0.0001). Receiver operating characteristic curves for the markers in combination in these two patient populations had an area under curve (AUC) of 0.79 in the training set and 0.8 in the test set. A CART (Classification and Regression Trees) model developed in the training set further improved the AUC in the test set to 0.898. CONCLUSIONS: Plasma markers, particularly when assessed in combination, can be used to monitor patients for disease recurrence and can compliment currently used lactate dehydrogenase and imaging studies; prospective validation is warranted.