An Oncogenic NTRK Fusion in a Patient with Soft-Tissue Sarcoma with Response to the Tropomyosin-Related Kinase Inhibitor LOXO-101

Abstract

Oncogenic TRK fusions induce cancer cell proliferation and engage critical cancer-related downstream signaling pathways. These TRK fusions occur rarely, but in a diverse spectrum of tumor histologies. LOXO-101 is an orally administered inhibitor of the TRK kinase and is highly selective only for the TRK family of receptors. Preclinical models of LOXO-101 using TRK-fusion-bearing human-derived cancer cell lines demonstrate inhibition of the fusion oncoprotein and cellular proliferation in vitro, and tumor growth in vivo. The tumor of a 41-year-old woman with soft-tissue sarcoma metastatic to the lung was found to harbor an LMNA-NTRK1 gene fusion encoding a functional LMNA-TRKA fusion oncoprotein as determined by an in situ proximity ligation assay. In a phase I study of LOXO-101 (ClinicalTrials.gov no. NCT02122913), this patient's tumors underwent rapid and substantial tumor regression, with an accompanying improvement in pulmonary dyspnea, oxygen saturation, and plasma tumor markers.

Significance: TRK fusions have been deemed putative oncogenic drivers, but their clinical significance remained unclear. A patient with a metastatic soft-tissue sarcoma with an LMNA-NTRK1 fusion had rapid and substantial tumor regression with a novel, highly selective TRK inhibitor, LOXO-101, providing the first clinical evidence of benefit from inhibiting TRK fusions.

Figure 1. LOXO-101 inhibition of cancer cells harboring oncogenic TRK

(A)–(C) Dose-dependent inhibition with LOXO-101 is demonstrated in three cancer cell line models of oncogenic TRK. CUTO-3.29 lung adenocarcinoma with MPRIP-NTRK1 (A), KM12 colorectal cancer with TPM3-NTRK1 (B), and MO-91 acute myeloid leukemia with ETV6-NTRK3 (C) cell lines were treated with a dose range of LOXO-101 and cellular proliferation was assayed by MTS assay. The cellular was IC₅₀ was ~59.4 ± 2.2 nM for CUTO-3.29, 3.5 ± 0.7 nM for KM12 and 1.0 ± 0.05 nM for MO-91. (D)–(F) Target inhibition and downstream signaling following LOXO-101 treatment in cancer cell line models of oncogenic TRK. (D) LOXO-101 inhibits phosphorylation of Y496 of the MPRIP-TRKA kinase and phosphorylation of T202/Y204 of ERK1/2 in CUTO-3.29 cells. (E) LOXO-101 inhibits phosphorylation of Y496 of TPM3-TRKA kinase and downstream phosphorylation of ERK1/2 and S473 AKT in KM12 cells. (F) LOXO-101 inhibits phosphorylation of TEL-TRKC kinase and ERK1/2 and AKT phosphorylation in MO-91 cells. Cells were treated for 2 hours with the indicated doses of LOXO-101 or DMSO alone and cell lysates were analyzed using the indicated antibodies except for MO-91 cells were treated for 2 hours with 100 nM of LOXO-101 or DMSO alone and cell lysates were immunoprecipitated with an anti-TRK antibody followed by immunoblot analysis with the indicated antibodies. (G) LOXO-101 inhibits tumor growth in a KM12 colorectal xenograft model. Percent changes from baseline tumor volume in nude mice (n = 10/group) injected subcutaneously with KM12 cells and treated with diluent (control), 60mg/kg/dose or 200mg/kg/dose daily for 14 days are shown. p-values for comparisons between the indicated treatment group and the control group are indicated as *, p < 0.05. and **, p-value < 0.01.

Figure 2. Molecular characterization of tumor sample

Multimodality testing demonstrating genomic, transcriptional, and functional (protein) evidence of LMNA-NTRK1 gene fusion in the patient’s tumor sample. (A) The LMNA-NTRK1 gene fusion was identified in the patient’s tumor sample by the FoundationOneHeme panel, joining the first two exons of LMNA (NM_170707) with exon 11–17 of NTRK1 (NM_002529). (B) NTRK1 break-apart FISH was performed as previously described (7) and demonstrates both paired green (5′ NTRK1) and red (3′ NTRK1) signals corresponding to the normal NTRK1 gene (yellow arrow). Isolated red signals (red arrows) are observed in tumor nuclei (stained blue with DAPI) indicative of a chromosomal deletion leading to an NTRK1 gene fusion. (C) Chromatograph of DNA sequencing of RT-PCR product using LMNA (5′) and NTRK1 (3′) primers indicating the fusion breakpoint between exon 2 of LMNA and exon 11 of NTRK1. (D) TRK-SHC1 proximity ligation assay demonstrates robust signaling in the tumor cells but weak signaling in the thick walled blood vessel. Nuclei are stained with DAPI (blue) and the red signals represent a positive PLA indicative of TRKA-SHC1 protein complexes. A blood vessel is indicated within the partial ellipse (dotted white line). (E) Adjacent tumor tissue section stained with hematoxylin and eosin indicating a thick-walled blood vessel (within partial ellipse indicated by dotted white line) and flanking tumor nuclei.

Figure 3. Radiologic response to LOXO-101

Computed tomography (CT) obtained following pre-operative chemotherapy and primary tumor resection with arrow indicating the presence of an 18mm right lung nodule 4 months prior to starting LOXO-101 (A), baseline imaging just prior to dosing with LOXO-101 on study (B), and following 1 cycle (28 days) (C), and 4 cycles (4 months) (D) of dosing with LOXO-101. The patient was observed to have metastatic disease only in the lungs and therefore the CT scan images show axial (top) and coronal (bottom) images focusing on the thoracic cavity. The images demonstrate an initial rapid disease progression (A–B, 13 week interval) followed by a marked tumor response with decreased size and/or resolution of the numerous pulmonary metastases (B–D, 4-week and 16-week intervals since baseline).