Druggable oncogene fusions in invasive mucinous lung adenocarcinoma

Abstract

Purpose: To identify druggable oncogenic fusions in invasive mucinous adenocarcinoma (IMA) of the lung, a malignant type of lung adenocarcinoma in which KRAS mutations frequently occur.

Experimental design: From an IMA cohort of 90 cases, consisting of 56 cases (62%) with KRAS mutations and 34 cases without (38%), we conducted whole-transcriptome sequencing of 32 IMAs, including 27 cases without KRAS mutations. We used the sequencing data to identify gene fusions, and then performed functional analyses of the fusion gene products.

Results: We identified oncogenic fusions that occurred mutually exclusively with KRAS mutations: CD74-NRG1, SLC3A2-NRG1, EZR-ERBB4, TRIM24-BRAF, and KIAA1468-RET. NRG1 fusions were present in 17.6% (6/34) of KRAS-negative IMAs. The CD74-NRG1 fusion activated HER2:HER3 signaling, whereas the EZR-ERBB4 and TRIM24-BRAF fusions constitutively activated the ERBB4 and BRAF kinases, respectively. Signaling pathway activation and fusion-induced anchorage-independent growth/tumorigenicity of NIH3T3 cells expressing these fusions were suppressed by tyrosine kinase inhibitors approved for clinical use.

Conclusions: Oncogenic fusions act as driver mutations in IMAs without KRAS mutations, and thus represent promising therapeutic targets for the treatment of such IMAs.

Figure 1.

Oncogenic fusions in invasive mucinous LDAC. A, schematic representations of the wild-type proteins (top rows of each section) followed by the fusion proteins identified in this study. The breakpoints for each variant are indicated by blue arrows. TM, transmembrane domain. Locations of putative cleavage sites in the NRG1 polypeptide are indicated by dashed green lines. B, detection of gene-fusion transcripts by RT-PCR. RT-PCR products for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown below. Six IMAs (T) positive for gene fusions are shown alongside their corresponding non-cancerous lung tissues (N); labels below the gel image indicate sample IDs (see Table 1). C, pie chart showing the fraction of IMAs that harbor the indicated driver mutations.

Figure 2.

Oncogenic properties of gene-fusion products. A, ERBB3 activation by CD74-NRG1 fusion, demonstrated using the EFM-19 cell system. ERBB3, ERBB2,AKT, and ERK phosphorylation were examined in EFM-19 (reporter) cells treated for 30minuteswith conditioned media from H1299 cells exogenously expressing CD74-NRG1 cDNA. Phosphorylation was suppressed by HER-TKIs. B, ERBB4 activation by EZR-ERBB4 fusion. Stably transduced NIH3T3 cells were serum-starved for 24 hours and treated for 2 hours with DMSO (vehicle control) or TKIs. Phosphorylation of ERBB4 and ERK was suppressed by ERBB4-TKIs. EZR-ERBB4 protein was detected using an antibody recognizing ERBB4 polypeptides retained in the fusion protein. C, BRAF activation by TRIM24-BRAF fusion. Stably transduced NIH3T3 cells were serum-starved for 24 hours and treated for 2 hours with DMSO or kinase inhibitors. ERK phosphorylation (activation) was suppressed by sorafenib, a kinase inhibitor targeting BRAF, as well as by U0126, a MEK inhibitor. TRIM24-BRAF protein was detected using an antibody recognizing BRAF polypeptides retained in the fusion protein. D–F, anchorage-independent growth of NIH3T3 cells expressing CD74-NRG1 (D), EZR-ERBB4 (E), or TRIM24-BRAF (F) cDNA, and suppression of this growth by kinase inhibitors. Mock-, CD74-NRG1-, EZR-ERBB4-, and TRIM24-BRAF–transduced NIH3T3 cells were seeded in soft agar with DMSO alone or kinase inhibitors. Colonies > 100 μm in diameter were counted after 14 days. Column graphs show mean numbers of colonies ± SEM.

Figure 3.

Tumorigenicity of NIH3T3 cells expressing ERZ-ERBB4 or TRIM24-BRAF fusion cDNAs. A, tumor growth in nude mice injected with NIH3T3 cells expressing empty vector, EZR-ERBB4 fusion, or TRIM24-BRAF fusion. Cells were resuspended with 50% Matrigel and injected into the right flank of nude mice. Tumor size was measured twice weekly for 5 weeks. Data are shown as mean ± SEM. B, representative tumors were photographed on day 21. The numbers in parentheses indicate the ratio of the number of mice with tumors to the number of mice receiving cell injection.