The TPM3-NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition

Abstract

The NTRK1 gene encodes Tropomyosin-related kinase A (TRKA), the high-affinity Nerve Growth Factor Receptor. NTRK1 was originally isolated from a colorectal carcinoma (CRC) sample as component of a somatic rearrangement (TPM3-NTRK1) resulting in expression of the oncogenic chimeric protein TPM3-TRKA, but there has been no subsequent report regarding the relevance of this oncogene in CRC. The KM12 human CRC cell line expresses the chimeric TPM3-TRKA protein and is hypersensitive to TRKA kinase inhibition. We report the detailed characterization of the TPM3-NTRK1 genomic rearrangement in KM12 cells and through a cellular screening approach, the identification of NMS-P626, a novel highly potent and selective TRKA inhibitor. NMS-P626 suppressed TPM3-TRKA phosphorylation and downstream signaling in KM12 cells and showed remarkable antitumor activity in mice bearing KM12 tumors. Finally, using quantitative reverse transcriptase PCR and immunohistochemistry (IHC) we identified the TPM3-NTRK1 rearrangement in a CRC clinical sample, therefore suggesting that this chromosomal translocation is indeed a low frequency recurring event in CRC and that such patients might benefit from therapy with TRKA kinase inhibitors.

Keywords: Colorectal cancer; Kinase inhibitor; NMS-P626; TPM3-NTRK1 rearrangement; TRKA.

Figure 1

NMS‐P626: Chemical structure and cellular activity A) Chemical structure of NMS‐P626, B) IC50 determination of NMS‐P626 activity against KM12 cell line, C) Antiproliferative activity of NMS‐P626 on a panel of 78 tumor cell lines, expressed as 1/IC50 in micromoles/liter (μM−1).

Figure 2

Genomic characterization of TPM3‐NTRK1 fusion in KM12 cell line A) End‐point PCR was performed on cDNA from KM12 cells with F/R primer couples designed to amplify regions in a) 1F/R: NTRK1 cytoplasmic domain, b) 2F/R: NTRK1 extracellular domain, c) 3F/R: TPM3‐NTRK1 rearrangement, d) 4F/R: TPM3 extracellular domain. See Supplementary Table S3 and Supplementary Figure S2 for primers sequence and position. B) Schematic representation of TPM3‐NTRK1 genomic DNA breakpoints in the KM12 cell line. The position of the breakpoints leading to the TPM3‐NTRK1 rearrangement is indicated with respect to genome reference hg19 version. Dark boxes: exons. Light boxes: introns. Striped box: alternative splicing exon in the TPM3 sequence.

Figure 3

Characterization by Western Blot analysis of the TPM3‐TRKA fusion protein and in vitro and in vivo mechanism of action of NMS‐P626 in the KM12 tumor model A) Anti‐TRKA‐immunoprecipitated proteins were subjected to Western Blot analysis using anti‐TRKA antibody (left panel) or anti‐TPM3 antibody (right panel). KM12: total cell lysates, C: control lane (lysis buffer), B) In vitro inhibition of TRKA phosphorylation and downstream signaling pathways in KM12 cells. Cells were treated for 2 h with increasing concentrations of NMS‐P626 and total protein lysates were analyzed by Western Blot with the indicated Ab, C) In vivo efficacy of NMS‐P626 in KM12 xenograft model. Nu/Nu mice bearing established KM12 xenografts were administered NMS‐P626 per os bid at the indicated doses or vehicle for 10 consecutive days, D) Ex‐vivo target modulation analysis. Nu/Nu mice bearing established KM12 xenografts were administered a single dose of 100 mg/kg NMS‐P626 per os and animals were sacrificed 6 h or 12 h after treatment. Tumors were resected, snap frozen and protein lysates were analyzed by Western Blot with the indicated Ab.

Figure 4

Identification of the TPM3‐NTRK1 rearrangement in a clinical sample RNA extracted and reverse transcribed from T27 colorectal cancer sample was analyzed for the presence of TPM3‐NTRK1 rearrangement using primers specifically designed across TPM3‐NTRK1 junction by A) RT‐qPCR (Supplementary Table S4, primers juncF/R) and B) end‐point PCR (Supplementary Table S3, primers 7F/R). We used the KM12 cell line as positive control and the K562 cell line expressing full‐length TRKA as negative control. T29 is a representative clinical sample showing no expression of the chimeric cDNA. C is the no template control.

Figure 5

IHC detection of the intracellular domain of TRKA IHC was performed using a commercial antibody recognizing the C‐terminal portion of TRKA (Epitomics). A–C) Surgical sample (T27) from right hemicolectomy with a diagnosis of poorly differentiated adenocarcinoma showing strong cytoplasmic immunohistochemical reactivity to anti‐TRKA. Some cells seem to show a membrane positivity that, indeed, is due to the presence of a scant cytoplasm (black arrows). A) 50× magnification; B) 100× magnification; C) 200× magnification D–E) Bioptic sample of the same tumor showing a strong immunoreactivity for TRKA antibody (bottom right). In the same figure normal colonic tissue (upper left) with no positivity is found D) 50× magnification, E) 200× magnification.