A large microRNA cluster on chromosome 19 is a transcriptional hallmark of WHO type A and AB thymomas

Abstract

Background: Thymomas are one of the most rarely diagnosed malignancies. To better understand its biology and to identify therapeutic targets, we performed next-generation RNA sequencing.

Methods: The RNA was sequenced from 13 thymic malignancies and 3 normal thymus glands. Validation of microRNA expression was performed on a separate set of 35 thymic malignancies. For cell-based studies, a thymoma cell line was used.

Results: Hierarchical clustering revealed 100% concordance between gene expression clusters and WHO subtype. A substantial differentiator was a large microRNA cluster on chr19q13.42 that was significantly overexpressed in all A and AB tumours and whose expression was virtually absent in the other thymomas and normal tissues. Overexpression of this microRNA cluster activates the PI3K/AKT/mTOR pathway. Treatment of a thymoma AB cell line with a panel of PI3K/AKT/mTOR inhibitors resulted in marked reduction of cell viability.

Conclusions: A large microRNA cluster on chr19q13.42 is a transcriptional hallmark of type A and AB thymomas. Furthermore, this cluster activates the PI3K pathway, suggesting the possible exploration of PI3K inhibitors in patients with these subtypes of tumour. This work has led to the initiation of a phase II clinical trial of PI3K inhibition in relapsed or refractory thymomas (https://ichgcp.net/clinical-trials-registry/NCT02220855).

Conflict of interest statement

M Radovich has received honoraria from Life Technologies Corporation. J Zhu is a former employee of Cofactor Genomics, LLC. J Glasscock is currently an employee, owner, and CEO of Cofactor Genomics, LLC.

Figures

Figure 1

Hierarchical clustering of thymoma samples. (A) Unsupervised hierarchical clustering of all expressed RefSeq genes in the thymoma and normal samples by RNA-Seq. Clustering was performed using RPKM values of expressed genes. The dendrogram demonstrates separation of the samples based on gene expression that is completely concordant with the WHO histological subtype (LR=lymphocyte rich). Red indicates higher expression and blue indicates lower expression. (B) Unsupervised hierarchical clustering of all expressed precursor microRNAs (as defined by the UCSC genome browser) in the thymoma and normal samples. The dendogram demonstrates a mixing of the A and AB thymomas with separation of the other subtypes.

Figure 2

Validation of expression of the C19MC cluster by qPCR in an independent sample set of 35 thymomas. Expression data demonstrate a separation of the samples into two distinct groups. All A and AB samples were in the C19MC-positive group, whereas the B1, B2, and B3 samples were in the C19MC-negative group, except for one discordant B1 sample in the C19MC-positive group. Two-digit numbers after the WHO subtype below the map are the sample numbers (from Supplementary Table 1). Six-digit values on the right side of the heatmap after the microRNA gene symbol represent the miRNA qPCR assay IDs from Life Technologies.

Figure 3

PI3K pathway analysis among thymoma subtypes. (A) Network analysis demonstrates overexpressed and underexpressed genes in A and AB thymomas vs B3 thymomas (FDR <25%) that would be expected of an overactivated PI3K/AKT pathway. Red indicates upregulated expression in A and AB thymomas and green indicates downregulated expression in A and AB thymomas. (B) Protein ELISA for activated phospho-AKT (Ser 473), and PTEN in a validation set of 35 thymomas and 3 normal tissues.

Figure 4

Functional analysis of key miRNAs within the C19MC cluster. The IU-TAB1 cell line was transfected with mir-517a, mir-519d, or the combination, along with a scrambled miRNA and mock transfected negative controls. The PTEN, phospho (p)-AKT (Ser 473), AKT (pan-AKT1/2/3), phospho-mTOR (Ser 2448), mTOR, phospho-p70 S6K (Ser 371), phospho-4E-BP1 (Thr 37/46), and β-actin protein levels were analysed using western blot.

Figure 5

The thymoma AB cell line, IU-TAB 1, was tested with increasing log-doses of various PI3K and dual PI3K/mTOR inhibitors currently in phase 2 or 3 clinical trial. Cells were treated for 72 h. The y axis represents percent change in cell viability. The dual PI3K/mTOR inhibitors, PF-04691502 and BEZ235, demonstrated the best reduction in cell viability at an IC50 of 118 and 210 nM, respectively.