Oncogenic and drug-sensitive NTRK1 rearrangements in lung cancer

Abstract

We identified new gene fusions in patients with lung cancer harboring the kinase domain of the NTRK1 gene that encodes the high-affinity nerve growth factor receptor (TRKA protein). Both the MPRIP-NTRK1 and CD74-NTRK1 fusions lead to constitutive TRKA kinase activity and are oncogenic. Treatment of cells expressing NTRK1 fusions with inhibitors of TRKA kinase activity inhibited autophosphorylation of TRKA and cell growth. Tumor samples from 3 of 91 patients with lung cancer (3.3%) without known oncogenic alterations assayed by next-generation sequencing or fluorescence in situ hybridization demonstrated evidence of NTRK1 gene fusions.

Figure 1. Discovery and validation of oncogenic NTRK1 gene fusions in lung cancer samples

(a) Schematic of genomic rearrangement from tumor samples harboring MPRIP-NTRK1 and CD74-NTRK1 using the FoundationOne Next Generation Sequencing Assay including chromosomal breakpoints for each gene rearrangement. (b) Break-apart FISH analysis of MPRIP-, CD74-, and unknown-NTRK1 tumor samples showing clear separation of green (5′) and red (3′) signals corresponding to the NTRK1 gene. (c) TRKA (NTRK1) fusions are autophosphorylated and activate key downstream signaling pathways. Representative immunoblot analyses (n = 3) of cell lysates from 293T cells expressing RIP-TRKA and CD74-TRKA, but not their kinase dead (KD) variants display phosphorylation of critical tyrosine residues and activation of pERK. TPM3-TRKA was expressed in 293T cells as a positive control. (d) NTRK1 fusions support cellular proliferation. MTS assay of Ba/F3 demonstrates that cells expressing RIP-TRKA, CD74-TRKA, EML4-ALK, or full length TRKA supplemented with NGF proliferate in the absence of IL-3, whereas Ba/F3 cells expressing EV or the kinase dead variant of RIP-TRKA do not proliferate (n = 3). Values represent the mean ± SEM. (e) MPRIP- or CD74-NTRK1 gene fusions induce tumorigenesis. NIH3T3 cells expressing RIP-TRKA, RIP-TRKA kinase dead (KD), CD74-TRKA, and EML4-ALK or empty vector were injected into the flanks of nude mice and observed for tumor growth. Representative pictures taken at day 12 following injection are shown. The numbers of tumors induced in the injected animals are shown in parentheses.

Figure 2. Drug treatment inhibits activation of TRKA, downstream signaling, and proliferation in cells expressing NTRK1 fusions

(a) RNAi knockdown of NTRK1 inhibits cell proliferation in a cell line harboring TPM3-NTRK1. KM12 cells were analyzed by MTS proliferation assay 1-5 days after siRNA transfection (left, n = 3). (b) Ba/F3 cells expressing MPRIP-NTRK1 (RIP-TRKA) or empty vector (EV) were lysed after 5h of treatment with the indicated doses of drugs (G = gefitinib 1000nM) or DMSO control (C). (c) CUTO-3 lung cancer cells harboring the MPRIP-NTRK1 gene fusion were treated with the indicated doses and drugs and subjected to immunoblot analysis. (d) Treatment of Ba/F3 cells expressing the MPRIP-TRKA fusion with TRKA inhibitors inhibits cell proliferation as measured by MTS assay (a-c, n = 5). Values represent the mean ± SEM. Ba/F3 cells expressing the MPRIP-TRKA fusion demonstrate inhibition of proliferation by the pan-TRK inhibitor, ARRY-470, and the multi-kinase inhibitor, CEP-701, but not the EGFR inhibitor, gefitinib.