Activating MET kinase rearrangements in melanoma and Spitz tumours

Abstract

Oncogenic gene fusions have been identified in many cancers and many serve as biomarkers or targets for therapy. Here we identify six different melanocytic tumours with genomic rearrangements of MET fusing the kinase domain of MET in-frame to six different N-terminal partners. These tumours lack activating mutations in other established melanoma oncogenes. We functionally characterize two of the identified fusion proteins (TRIM4-MET and ZKSCAN1-MET) and find that they constitutively activate the mitogen-activated protein kinase (MAPK), phosphoinositol-3 kinase (PI3K) and phospholipase C gamma 1 (PLCγ1) pathways. The MET inhibitors cabozantinib (FDA-approved for progressive medullary thyroid cancer) and PF-04217903 block their activity at nanomolar concentrations. MET fusion kinases thus provide a potential therapeutic target for a rare subset of melanoma for which currently no targeted therapeutic options currently exist.

Figure 1. Spitzoid Melanoma with ZKSCAN1-MET fusion

(a) Histopathology (4x, scale bar, 1 mm) showing a dome-shaped nodule with a thickened epidermis and sheets of spindled and epithelioid melanocytes in the dermis, some with central necrosis (20x, arrow, scale bar, 200 μm). High power view (20x, lower panel) demonstrates small nests of melanocytes in infiltrative array and a mitotic figure (arrow) in the deep portion of the tumor. Scale bar, 25 μm. (b) aCGH profile (top panel) with copy number increases involving chromosomes 7 and 12p. Chromosome 7 shows amplification of multiple regions on the long arm, including the 5’ end of ZKSCAN1 (red) and the 3’ end of MET (blue) (lower panels). (c) Stacks of sequencing reads from Integrated Genome Viewer spanning the breakpoints, with unaligned portions (rainbow-colored) flanking one side of the fusion junction in ZKSCAN1 (left) and MET (right). Reads whose mate pair maps to the opposite side of the fusion junction are maroon. The DNA sequence of the fusion junction is displayed, with bases that map to ZKSCAN1 in red, and those that map to MET in blue with their corresponding gene models above. Two bases of microhomology at the fusion junction are displayed in purple. The exonic structure of the predicted fusion transcript is shown with the sequencing trace from RT-PCR confirming its expression beneath.

Figure 2. Reciprocal translocation resulting in LRRFIP1-MET fusion in an atypical Spitz tumor

(a) Histopathology showing large nests of epithelioid melanocytes with pericellular clefting (4x, left panel, Scale bar, 300 μm) and scattered mitoses (20x, right panel, arrow, Scale bar, 50 μm) in the papillary dermis under a thickened epidermis. (b) Inferred succession of genetic events with supporting genomic data. Left panel: Reciprocal translocation between chromosomes 2 and 7 generates two derivative chromosomes with LRRFIP1-MET and MET-LRRFIP1 fusion genes, respectively. Chimeric reads supporting the fusion junctions are mapped to intron 14 of MET with the rainbow colored portion of the reads mapping to chromosome 2. Right panel: Subsequent homologous recombination of the derivative chromosome 2 results in a region of copy number neutral loss of heterozygosity on 2q and gain of the LRRFIP1-MET fusion gene. (c) Copy number and major allele frequency plots for chromosomes 2 and 7 demonstrate loss of 2q and gain of 7q distal to the breakpoints and allelic imbalance on 2q. (d) The exonic structure of the predicted fusion transcript with the sequencing trace from RT-PCR confirming its expression. (e) Strong diffuse expression of the kinase domain of MET is demonstrated by immunohistochemistry, with moderate staining for p-MET (4x, Scale bar, 100 μm).

Figure 3. Domain structure of MET Fusions

The portion from the N-terminal partner is displayed in pink with multimerization domains highlighted in red (CC= coiled coil domain, SCAN= SCAN domain). The MET portion (starting from exon 15 in all cases) is colored blue with the kinase domain highlighted in dark blue.

Figure 4. MET fusions constitutively activate oncogenic signaling pathways and are inhibited by cabozantinib and PF-04217903

(a) Melan-a cells (immortalized mouse melanocytes) were stably transduced with full length MET, TRIM4-MET and ZKSCAN1-MET expression constructs. The expressed fusion proteins were detected with by MET antibody at the predicted molecular weights of 79 and 65 kDa (longer exposure upper MET panel, shorter exposure lower MET panel). TRIM4-MET and ZKSCAN1-MET were phosphorylated at tyrosine 1234/1235 in the absence of serum. (b) Treatment with cabozantinib or PF-04217903 for four hours (both at 100 nM) significantly decreased p-MET and downstream signaling.

Figure 5. ZKSCAN-1 MET is tumorigenic

(a) Melan-a stably transduced with a ZKSCAN1-MET expression construct were injected into NOD/SCID/interleukin 2 receptor γ null mice (1.5 million cells, bilateral flank injections) with matched GFP transduced controls. All six injection sites with ZKSCAN1-MET expressing melan-a cells developed tumors within 5 weeks (black circles), whereas no tumors developed at the sites injected with GFP transduced controls (n=6) after 12 weeks (blue triangles). (b) Tumor formation 28 days after injection with ZKSCAN1-MET expressing melan-a cells. Scale bar, 0.5 cm. (c) Histopathology of ZKSCAN1-MET tumor (4x) demonstrates tumor cells diffusely within the skin and subcutaneous tissue. Scale bar, 500 μm. (d) At high power (20x) tumor cells have hyperchromatic and irregularly sized nuclei and mitoses are present. Scale bar, 10 μm. (e) Tumor cells have variable positivity for the kinase domain of MET (20x). Scale bar, 10 μm. (f) Tumor cells have variable levels of p-MET positivity (20x). Scale bar, 10 μm.