Discovery and functional characterization of the oncogenicity and targetability of a novel NOTCH1-ROS1 gene fusion in pediatric angiosarcoma

Abstract

Angiosarcomas are rare, malignant soft tissue tumors in children that arise in a wide range of anatomical locations and have limited targeted therapies available. Here, we report a rare case of a pediatric angiosarcoma (pAS) with Li-Fraumeni syndrome (LFS) expressing a novel NOTCH1-ROS1 gene fusion. Although both NOTCH1 and ROS1 are established proto-oncogenes, our study is the first to describe the mechanistic role of NOTCH1-ROS1 fusion arising via intrachromosomal rearrangement. NOTCH1-ROS1 displayed potent neoplastic transformation propensity in vitro, and harbors tumorigenic potential in vivo, where it induced oncogenic activation of the MAPK, PI3K/mTOR, and JAK-STAT signaling pathways in a murine allograft model. We found an unexpected contribution of the NOTCH1 extracellular region in mediating NOTCH1-ROS1 activation and oncogenic function, highlighting the contribution of both NOTCH1 and ROS1 fusion partners in driving tumorigenicity. Interestingly, neither membrane localization nor fusion protein dimerization were found to be essential for NOTCH1-ROS1 fusion oncogenicity. To target NOTCH1-ROS1-driven tumors, we tested both NOTCH1-directed inhibitors and ROS1-targeted tyrosine kinase inhibitors (TKI) in heterologous models (NIH3T3, Ba/F3). Although NOTCH1 inhibitors did not suppress NOTCH1-ROS1-driven oncogenic growth, we found that oral entrectinib treatment effectively suppressed the growth of NOTCH-ROS1-driven tumors. Taken together, we report the first known pAS case with a novel NOTCH1-ROS1 alteration along with a detailed report on the function and therapeutic targeting of NOTCH1-ROS1. Our study highlights the importance of genomic profiling of rare cancers such as pAS to reveal actionable drivers and improve patient outcomes.

Keywords: metastatic angiosarcoma.

Figure 1.

Histological and sequencing characteristics of a pediatric angiosarcoma with NOTCH1–ROS1 fusion that drives oncogenic cellular phenotypes. (A) A biopsy of the scapula 2 yr previous show a low-grade vascular lesion consisting of individual muscle fibers wrapped by a single layer of endothelial cells (hematoxylin and eosin [H&E], top panel) that are positive for lymphatic marker D240 (middle panel). The bottom panel shows 6 mo after radiation, incisional biopsy revealed a proliferation of malignant cells forming irregular vascular channels, consistent with angiosarcoma. All histology images are 20×. (B) Targeted RNA-seq results showing multiple next-generation sequencing (NGS) reads spanning the breakpoint (top) with confirmatory Sanger sequencing of cDNA exhibiting breakpoint (bottom). (C) NOTCH1–ROS1 fusion protein retains: NOTCH1 exons 1–30 encoding epidermal growth factor (EGF) domains 1–36, LNR 1–3, and transmembrane domain. ROS1 exons 34–43 encoding the transmembrane domain and complete tyrosine kinase. (D) Soft agar colony assays with stable NIH3T3 cell lines, quantification of colony counts shown for n = 5. (E) Ba/F3 proliferation assay performed in the absence of IL-3. (F) Flank xenograft tumor measurements of NSG mice engrafted with stable NIH3T3 cell lines, n = 5, P < 0.05.

Figure 2.

Activated ROS1 kinase in NOTCH1–ROS1 drives downstream signaling via MAPK, PI3K/Mtor, and JAK–STAT pathways. Western blot analysis of (A) stable NIH3T3 and (B) Ba/F3 cell lines showing phosphorylated (p-) and total (t-) protein levels of MAPK, PI3K/Mtor, and JAK–STAT pathways. Western blot analysis to show activation of ROS1 kinase in (C) stable NIH3T3 and (D) Ba/F3 cell lines showing phosphorylated (p-) and total (t-) protein levels. High exposure tROS1 blot is included in Supplemental Figure 1a to show ROS1 levels in ROS1 NIH3T3 cell model.

Figure 3.

Truncation of NOTCH1 extracellular domains causes loss of NOTCH1–ROS1 oncogenicity, despite lack of efficacy with existing preclinical NOTCH1 inhibitors. (A) Diagrammatic representation of NOTCH1, ROS1, NOTCH1–ROS1, and Trunc. NOTCH1–ROS1 predicted protein domains and localization. (B) Soft agar colony assays with stable NIH3T3 cells, quantification of colony counts shown for n = 5. (C) Ba/F3 proliferation assay performed in the absence of IL-3. Western blot analysis of (D) stable NIH3T3 and (E) Ba/F3 cell lines showing phosphorylated (p-) and total (t-) protein levels of MAPK, PI3K/mTOR, JAK–STAT pathways, and activation level of ROS1 kinase. fx1 represents NOTCH1–ROS1 (256 kDa); ◄ represents Trunc. NOTCH1–ROS1 (118 kDa); fx2 represents Cleaved NOTCH1–ROS1 (∼75 kDa). (F,G) Soft agar colony formation analysis with NIH3T3 cell models in presence of increasing doses of (F) gamma secretase inhibitor, RO4929097, and (G) ADAM 10/17 inhibitor, GW280264×. n = 10 each.

Figure 4.

ROS1-directed targeted inhibitors target NOTCH1–ROS1-driven signaling and in vitro and in vivo oncogenic growth. (A) Ba/F3 proliferation assay using clinical ROS1-directed inhibitors in BA/F3 cell growth. (B) Soft agar colony formation analysis with NIH3T3 cell models in presence of increasing doses of entrectinib and crizotinib. n = 10, * denotes P-value ≤0.05 and ** denotes P-value ≤0.01. Western blot analysis of (C) Ba/F3 model and (D) NIH3T3 model upon treatment with crizotinib and entrectinib. (E) Tumor volume plot from in vivo flank xenograft of NOTCH1–ROS1 expressing NIH3T3 and vector control cells in NSG mice and bi-daily oral gavage treatment with entrectinib. n = 8 Significant decrease compared to * NOTCH1–ROS1 + Vehicle, ** NOTCH1–ROS1 + 15 mg/kg Entrectinib, *** NOTCH1–ROS1 + 30 mg/kg Entrectinib. (F) Western blot analysis of mouse tumor lysates to show targeting of MAPK, PI3K, and JAK/STAT and expression of fusion protein in the myc-tag blot.