Neurotrophic tyrosine kinases (NTRKs), including NTRK1, NTRK2 and NTRK3, are transmembrane tyrosine kinase receptors involved in central and peripheral nervous system development. NTRK gene fusions are implicated in a broad range of cancers.
Under normal conditions, NTRK activation by neurotrophin binding stimulates homodimerization, autophosphorylation and subsequent signaling through the phosphoinositide 3-kinase (PI3K), AKT and mitogen-activated protein kinase (MAPK) pathways. This promotes neuronal cell survival and neuronal subtype differentiation, in addition to sustained synaptic activity and neuron growth throughout adulthood (7). Originally characterized as oncogenic fusions with tropomyosin in colon cancer (1,2) and thyroid carcinomas (3-5), NTRKs were later discovered to be receptors for nerve growth factors (NGFs) as well (6).
Increasing amounts of tumor sequencing studies highlight the broad cancer-types associated with NTRK gene fusions (shown per gene, below). Many oncogenic NTRK translocations and mutations result in constitutive activation of tyrosine kinase domains. Given the recent idenitification of selective tyrosine kinase inhibitors, this is of particular interest to researchers (8-10). Targeted tools, such as Archer FusionPlex assays, can assist researchers in identification and molecular characterization of novel and known fusions of NTRK genes with significantly lower cost than whole-genome or RNA-seq based approaches.
The NTRK1 gene, located on human chromosome 1q23.1, encodes the neurotrophic tyrosine kinase receptor-1, known as TrkA, and shares 47% nucleic acid identity with NTRK2 and NTRK3. TrkA was discovered and characterized as a theretofore unknown tyrosine kinase fused to tropomyosin (TPM3) in colon carcinoma (1,20) and mutated in thyroid carcinomas (3-5). Subsequent studies revealed TrkA as the high-affinity receptor for NGF (6), though TrkA also binds neurotrophin-3 (NT3) with lower affinity (11). The binding of NGF or NT3 enables TrkA dimerization and autophosphorylation at multiple tyrosine residues. These phosphorylations facilitate binding of Shc, phospholipase-Cγ (PLCγ), and protein adapters containing phosphotyrosine-binding (PTB) domains resulting in activation of the Ras/MAPK, PI3K, AKT and PLCγ pathways (7,21-23).
Three isoforms of NTRK1 are characterized in the literature: TrkA-I is found in non-neuronal tissues, TrkA-II in neuronal cell types (24) and TrkA-III in undifferentiated early neural progenitors and neural crest progenitor cells (25). TrkA-III excludes NTRK1 exons 6, 7 and 9 producing a gene product missing several extracellular domains (25). The TrkA-II isoform also excludes the extracellular IG-C1 and N-glycosylation domains, is unresponsive to NGF and may constitutively activate the IP3K, AKT1 and NFkB pathways (25).
NTRK1 gene fusions are characterized in numerous cancer types. These cancers include intrahepatic cholangiocarcinoma by RABGAP1L-NTRK1 fusion (12), pediatric high-grade glioma with TP53-NTRK1 fusion (14), glioblastoma multiforme brain tumors from BCAN-NTRK1 and NFASC-NTRK1 fusions (15), LMNA-NTRK1 translocation in Spitz tumors and spitzoid melanomas (17), various lung cancers from MPRIP-NTRK1 and CD74-NTRK1 fusions (16), colon carcinoma with TPM3-NTRK1 (1) and papillary thyroid carcinoma (4). The pan-cancer nature of NTRK1 translocations supports the idea that miscreant tyrosine kinase function contributes to transformative phenotypes. This cancer-causing potential may arise through upregulation of the aberrant tyrosine kinase domains (15) or through stimulating dimerization via novel translocated extracellular domains non-responsive to NGF stimulation (16,17).
NTRK2 (also known as TrkB) is the high-affinity receptor for brain-derived neurotrophic factor (BDNF), though NTRK2 also binds neurotrophin-4 (NT4) (26-30) and neurotrophin-3 (NT3) with diminished affinity (26,29). Though principally localized to central and peripheral nervous system tissues (26), isoform TrkB-T1 is also detected in the heart, kidneys and pancreas (31). Extracellular ligand-binding induces autophosphorylation (32), promoting survival and neurodifferentiation (33) and mediation of synaptic plasticity (34,35). NTRK2 exhibits great isoform complexity containing 24 exons (36) on chromosome 9 (37) producing at least 36 isoforms (31).
NTRK2 fusions contributing to human cancer have recently been detected. RNA-seq data from 7000 samples derived from The Cancer Genome Atlas uncovered previously characterized and novel NTRK2 fusions in low-grade gliomas (AFAP1-NTRK2), head and neck squamous cell carcinoma (PAN3-NTRK2) and lung adenocarcinoma (TRIM24-NTRK2) (18). A 2013 next-generation sequencing study of 96 pilocytic astrocytomas identified constitutively active kinase activity from novel NACC2:NTRK2 and QKI:NTRK2 fusions (19) in 3 of the 9 non-cerebellar tumors lacking other identified fusions. NTRK2 translocations also are oncogenic in neuroblastoma (38), ovarian, pancreatic, prostate, hepatocellular and gastric cancers (reviewed in (39)).
NTRK3 was discovered in hippocampal cerebral cortex and granular cell layers of the cerebellum and binds NT3 but not BDNF or NGF (40). Subsequent analysis implied a role for NTRK3 in oncogenesis since high-level NTRK3 mRNA expression strongly correlates with challenging disease progression in medulloblastoma patients (41). NTRK3 translocations are characterized across many cancer types, including radiation-associated thyroid cancer (42), high grade gliomas (14), acute myeloid leukemia (43-45), ductal carcinoma (46-49), fibrosarcomas (50-52), congenital mesoblastic nephroma (53) and secretory breast carcinoma (54).
Of the relatively few gene fusions characterized with NTRK, translocation of NTRK3 and Ets family transcription factor 6 (ETV6) appears with the greatest frequency (42-44,46-54). This fusion imparts the N-terminal pointed (PNT) domain mediating auto-oligomerization from ETV6 to the kinase domain of NTRK3, contributing to its autoactivation and transforming activity (45,55-57).
Thyroid malignancies are strongly skewed towards papillary thyroid carcinomas (PTC), which accunt for ~80% of all cases (58). Seventy percent of PTC cases result from rearrangements of RET or NTRK and point mutations in RAS or BRAF (58). NTRK translocations generally result in constitutively active tyrosine kinase activity upregulating the Ras-Raf1-Mek1-Erk1/2 or PI3K-AKT pathways (16,59). These in turn converge on the MAPK pathway, of which mutations in a single component may be sufficient to yield a malignant phenotype (60). Thus, targeting NTRK fusions is an appealing pharmacological possibility actively pursued by current research investigators.
Ongoing research studies address NTRK tyrosine kinase activity inhibition as a potential therapeutic target. Tyrosine kinase inhibitors ARRY-470, CEP-701, CEP-751 and crizotinib were shown to reduce NTRK autophosphorylation and downstream phosphorylation of AKT and ERK in Ba/F3 cells containing TFG-NTRK1 translocations, and may more broadly affect other NTRK family members (16,61). CEP-701 is an orally active K252a analog in clinical evaluation for inhibition of tyrosine kinase receptors, suggested to be a potent tyrosine kinase inhibitor (9,10). CEP-751 is an indolocarbozale derivative which modest growth inhibition effects against NTRK2-overexpressing tumors (62). ARRY-470 is a small-molecule NTRK1 inhibitor of nanomolar sensitivity originally employed to attenuate cancer pain (63). CEP-751 and ARRY-470 were shown more effective inhibitors of NTRK1 than crizotinib in lung cancer clinical samples (64,65). Orally administrable AZ-23 has also shown early success at selectively inhibiting NTRK1 and NTRK2 (66), and selectively inhibited chimeric LMNA-NTRK1 autophosphorylation and activation of downstream AKT, ERK, S6 and PLCγ pathways in melan-a cells (17).
Given these successes, it comes as no surprise that several clinical trials of TRK inhibitors have been initiated to target aberrant NTRK tyrosine kinase activity, including LOXO-101 (TRKA/B/C; NCT02122913) and Ignyta's phase II clinical trial of entrectinib, RXDX-101 (TRKA/B/C, ALK, ROS1; NCT02097810), both specifically targeting NTRK gene-fusion positive non-small cell lung cancer (NSCLC) patients (for a full list, see ClinicalTrials.gov: http://clinicaltrials.gov/ct2/results?term=TRKA&Search=Search). The advent of these potent NTRK inhibitors and the realization that NTRK translocations are found in increasing types of cancers highlights the need for ongoing research to detect and molecularly characterize NTRK1 translocations to promote future treatment options.
|Fusion Partner||Disease||PubMed Evidence|
|RABGAP1L-NTRK1||Intrahepatic cholangiocarcinoma (ICC)||12|
|TPM3-NTRK1||Papillary thyroid carcinomas||4,68|
|PAN3-NTRK2||Head and neck squamous cell carcinoma||18|
|ETV6-NTRK3||Secretory breast carcinoma||54|
|ETV6-NTRK3||Congenital mesoblastic nephroma||53|
|ETV6-NTRK3||Radiation-associated thyroid cancer||42|
|ETV6-NTRK3||Acute myeloid leukemia (AML)||43,44,52|
Historically, NTRK fusions were discovered by cloning biologically active cDNAs from cell lines (1, 20, 26, 40). In the case of human samples, where both fusion partners are known, RT-PCR and qPCR probes can be generated to locate a predetermined fusion product (67). Fluorescence in-situ hybridization (FISH) assays, including fusion and break-apart probes, may subjectively describe the presence or absence of a fusion (46, 65).
Despite the plethora of methods used to detect gene translocations, discovery of novel partners using these methods is laborious, typically relying on primer-walking Sanger sequencing or whole exome sequencing. FISH assays require subjective analysis by trained experts, yet equivocal results are still common since processing slides may mechanically separate chromosomal regions betwixt different curls of the same sample. Varying degrees of natural chromosomal condensation may affect how far apart FISH probes hybridize, making fluorescent signals ambiguous. FISH, RT-PCR, and qPCR are all plagued by the common weakness of requiring very specific fusion locations in order to produce a result. Modern fusion-detection research relies heavily on next-generations sequencing technologies like whole genome sequencing, exome sequencing and RNA-seq to locate novel fusions (14, 16, 18, 19, 64). These studies are laborious considering the magnitude of data accumulated for each research sample, and require significant bioinformatics resources to unravel.
Targeted sequencing technologies hold promise for reducing the amount of data to interpret, but are challenged by the same caveat of RT-PCR and qPCR, namely the exact fusion loci must be known to generate primers for targeting. Archer's Anchored Multiplex PCR (AMP™) technology was designed to overcome this by using half-functional Y-adapters of known sequence which are ligated to next-generation sequence libraries upstream of any polymerase chain reactions. In cases where novel fusions are present, a gene-specific primer (GSP) to NTRKs can amplify against a known adapter sequence, producing sequencing-ready targeted libraries under circumstances that previously would require whole-genome, exome or RNA-seq to identify. Archer FusionPlex kits utilize AMP fusion detection technology, a simple lyophilized workflow and a purpose-built analysis pipeline to provide comprehensive fusion identification even without prior knowledge of fusion partners or breakpoints.
Although the list of TKIs being studied continues to grow, research is still needed to identify novel fusion partners and understand their mechanisms of action. Fortunately, through the widespread adoption of NGS, research on genetic abnormalities in cancer has significantly accelerated. NGS-based fusion detection has recently benefited from advancements like AMP that allows for discovery of novel fusions in a scalable manner, while bioinformatics tools have helped to convert that information into usable data. Through the clever applications of these types of tools, the cancer research community is poised to make groundbreaking discoveries to further understand the biology and targeting of genes such as the NTRK gene family.
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