Patients with advanced neuroblastoma (NB) receive multimodal clinical therapy, including the potent anthracycline chemotherapy drug doxorubicin (Dox). The acquisition of Dox resistance, however, is a major barrier to a sustained response and leads to a poor prognosis in advanced disease states, reinforcing the need to identify and inhibit Dox resistance mechanisms. In this context, we report on the identification and inhibition of a novel Dox resistance mechanism. This mechanism is characterized by the Dox-induced activation of the oncogenic TrkAIII alternative splice variant, resulting in increased Dox resistance, and is blocked by lestaurtinib, entrectinib, and crizotinib tyrosine kinase and LY294002 IP3-K inhibitors. Using time lapse live cell imaging, conventional and co-immunoprecipitation Western blots, RT-PCR, and inhibitor studies, we report that the Dox-induced TrkAIII activation correlates with proliferation inhibition and is CDK1- and Ca²⁺-uniporter-independent. It is mediated by ryanodine receptors; involves Ca²⁺-dependent interactions between TrkAIII, calmodulin and Hsp90; requires oxygen and oxidation; occurs within assembled ERGICs; and does not occur with fully spliced TrkA. The inhibitory effects of lestaurtinib, entrectinib, crizotinib, and LY294002 on the Dox-induced TrkAIII and Akt phosphorylation and resistance confirm roles for TrkAIII and IP3-K consistent with Dox-induced, TrkAIII-mediated pro-survival IP3K/Akt signaling. This mechanism has the potential to select resistant dormant TrkAIII-expressing NB cells, supporting the use of Trk inhibitors during Dox therapy in TrkAIII-expressing NBs.

Pediatric neuroblastomas (NBs) are heterogeneous, aggressive, therapy-resistant embryonal tumors that originate from cells of neural crest origin committed to the sympathoadrenal progenitor cell lineage. Stress- and drug-resistance mechanisms drive post-therapeutic relapse and metastatic progression, the characterization and inhibition of which are major goals in improving therapeutic responses. Stress- and drug-resistance mechanisms in NBs include alternative TrkAIII splicing of the neurotrophin receptor tropomyosin-related kinase A (NTRK1/TrkA), which correlates with post-therapeutic relapse and advanced-stage metastatic disease. The TrkAIII receptor variant exerts oncogenic activity in NB models by mechanisms that include stress-induced mitochondrial importation and activation. In this study, we characterize novel targetable and non-targetable participants in this pro-survival mechanism in TrkAIII-expressing SH-SY5Y NB cells, using dithiothreitol (DTT) as an activator and a variety of inhibitors by regular and immunoprecipitation Western blotting of purified mitochondria and IncuCyte cytotoxicity assays. We report that stress-induced TrkAIII misfolding initiates this mechanism, resulting in Grp78, Ca²⁺-calmodulin, adenosine ribosylating factor (Arf) and Hsp90-regulated mitochondrial importation. TrkAIII imported into inner mitochondrial membranes is cleaved by Omi/high temperature requirement protein A2 (HtrA2) then activated by a mechanism dependent upon calmodulin kinase II (CaMKII), alpha serine/threonine kinase (Akt), mitochondrial Ca²⁺ uniporter and reactive oxygen species (ROS), involving inhibitory mitochondrial protein tyrosine phosphatase (PTPase) oxidation, resulting in phosphoinositide 3 kinase (PI3K) activation of mitochondrial Akt, which enhances stress resistance. This novel pro-survival function for misfolded TrkAIII mitigates the cytotoxicity of mitochondrial Ca²⁺ homeostasis disrupted during integrated stress responses, and is prevented by clinically approved Trk and Akt inhibitors and also by inhibitors of 78kDa glucose regulated protein (Grp78), heat shock protein 90 (Hsp90), Ca²⁺-calmodulin and PI3K. This identifies Grp78, Ca²⁺-calmodulin, Hsp90, PI3K and Akt as novel targetable participants in this mechanism, in addition to TrkAIII, the inhibition of which has the potential to enhance the stress-induced elimination of TrkAIII-expressing NB cells, with the potential to improve therapeutic outcomes in NBs that exhibit TrkAIII expression and activation.

Pituitary neuroendocrine tumors (PitNETs) are generally benign but comprise an aggressive, invasive, therapy-resistant, metastatic subset, underpinning a need for novel therapeutic targets. PitNETs exhibit low mutation rates but are associated with conditions linked to alternative splicing, an alternative oncogene pathway activation mechanism. PitNETs express the neurotrophin receptor TrkA, which exhibits oncogenic alternative TrkAIII splicing in other neuroendocrine tumors. We, therefore, assessed whether TrkAIII splicing represents a potential oncogenic participant in PitNETs. TrkAIII splicing was RT-PCR assessed in 53 PitNETs and TrkA isoform(s) expression and activation were assessed by confocal immunofluorescence. TrkAIII splicing was also compared to HIF1α, HIF2α, SF3B1, SRSF2, U2AF1, and JCPyV large T antigen mRNA expression, Xbp1 splicing, and SF3B1 mutation. TrkAIII splicing was detected in all invasive and most non-invasive PitNETs and was significantly elevated in invasive cases. In PitNET lineages, TrkAIII splicing was significantly elevated in invasive PIT1 PitNETs and high in invasive and non-invasive SF1 and TPIT lineages. Immunoreactivity consistent with TrkAIII activation characterized PitNET expressing TrkAIII mRNA, and invasive Pit1 PitNETs exhibited elevated HIF2α expression. TrkAIII splicing did not associate with SF3B1 mutations, altered SF3B1, SRSF2, and U2AF1 or JCPyV large T antigen expression, or Xbp1 splicing. Therefore, TrkAIII splicing is common in PitNETs, is elevated in invasive, especially PIT1 tumors, can result in intracellular TrkAIII activation, and may involve hypoxia. The data support a role for TrkAIII splicing in PitNET pathogenesis and progression and identify TrkAIII as a novel potential target in refractory PitNETs.

Post-therapeutic relapse, poor survival rates and increasing incidence justify the search for novel therapeutic targets and strategies in cutaneous malignant melanoma (CMM). Within this context, a potential oncogenic role for TrkA in CMM is suggested by reports of NTRK1 amplification, enhanced TrkA expression and intracellular TrkA activation associated with poor prognosis. TrkA, however, exhibits tumour-suppressing properties in melanoma cell lines and has recently been reported not to be associated with CMM progression. To better understand these contradictions, we present the first analysis of potential oncogenic alternative TrkA mRNA splicing, associated with TrkA immunoreactivity, in CMMs, and compare the behaviour of fully spliced TrkA and the alternative TrkAIII splice variant in BRAF(V600E)-mutated A375 melanoma cells. Alternative TrkA splicing in CMMs was associated with unfolded protein response (UPR) activation. Of the several alternative TrkA mRNA splice variants detected, TrkAIII was the only variant with an open reading frame and, therefore, oncogenic potential. TrkAIII expression was more frequent in metastatic CMMs, predominated over fully spliced TrkA mRNA expression in ≈50% and was invariably linked to intracellular phosphorylated TrkA immunoreactivity. Phosphorylated TrkA species resembling TrkAIII were also detected in metastatic CMM extracts. In A375 cells, reductive stress induced UPR activation and promoted TrkAIII expression and, in transient transfectants, promoted TrkAIII and Akt phosphorylation, enhancing resistance to reductive stress-induced death, which was prevented by lestaurtinib and entrectinib. In contrast, fully spliced TrkA was dysfunctional in A375 cells. The data identify fully spliced TrkA dysfunction as a novel mechanism for reducing melanoma suppression, support a causal relationship between reductive stress, UPR activation, alternative TrkAIII splicing and TrkAIII activation and characterise a targetable oncogenic pro-survival role for TrkAIII in CMM.

Background: Alternative NTRK1/TrkA splicing resulting in TrkAIII expression, originally discovered in advanced-stage metastatic neuroblastomas, is also pronounced in prostate, medullary thyroid, glioblastoma multiforme, MCPyV-positive Merkel cell, cutaneous malignant melanoma, and pituitary neuroendocrine tumor subsets. In tumor models, TrkAIII exhibits actionable oncogenic activity equivalent to the TrkT3-fused oncogene, and in tumor cell lines, alternative TrkAIII splicing is promoted by hypoxia, nutrient deprivation, endoplasmic reticulum stress, and SV40 large T antigen, implicating tumor microenvironmental conditions and oncogenic polyoma viruses in tumor-associated TrkAIII expression. Collectively, these observations characterize TrkAIII as a potentially frequent, actionable oncogenic alternative to TrkA gene fusion in different tumor types. Currently, therapeutic approval for efficacious Trk inhibitors is restricted to Trk-fused gene positive tumors and not for tumors potentially driven by TrkAIII. Methods: With the therapeutically relevant aim of improving the identification of tumors potentially driven by TrkAIII, we have developed a TaqMan-based qRT-PCR assay for evaluating TrkAIII expression in tumor cDNAs. Results: This assay, validated using gel-purified fs-TrkA and TrkAIII cDNAs alone and in complex cDNA mixtures, employs primers and probes designed from fs-TrkA and TrkAIII sequences, with specificity provided by a TaqMan probe spanning the TrkAIII exon 5–8 splice junction. It is highly efficient, reproducible, and specific and can detect as few as 10 TrkAIII copies in complex RNAs extracted from either fresh or FFPE tumor tissues. Conclusions: Inclusion of this assay into precision oncology algorithms, when paired with fs-TrkA qRT-PCR and TrkA immune histochemistry, will make it easier to identify patients with therapy-resistant, advanced-stage metastatic Trk-fused gene-negative tumors potentially driven by TrkAIII, for whom approval of third-line effective Trk inhibitors could be extended.

PitNETs, although typically benign, comprise a small percentage of therapy-resistant aggressive, occasionally metastatic tumors, underpinning a need to identify novel therapeutic targets. PitNETs carry few mutations but associate with conditions that promote alternative splicing as an oncogenic alternative and express the TrkA neurotrophin receptor, which exhibits oncogenic alternative TrkAIII splicing in other tumors. Alternative TrkAIII splicing was assessed by RT-PCR in 53 PitNETs and compared to HIF1a, HIF2a, SF3B1, SRSF2, U2AF1 and JCPyV large T antigen mRNA expression, unconventional Xbp1 splicing, SF3B1 mutation, and TrkA isoform(s) immunoreactivity by confocal immunofluorescence (IF). Alternative TrkAIII mRNA splicing was detected in all invasive PitNET phenotypes, was significantly elevated in invasive compared to non-invasive PIT1 PitNETs, relatively high in invasive and non-invasive SF1 and TPIT PitNETs, and was associated with IF consistent with intracellular TrkAIII activation but not with hotspot SF3B1 mutations, altered SF3B1, SRSF2 and U2AF1 splice factor mRNA expression, JCPyV large T antigen expression or unconventional Xbp1 splicing. A potential role for hypoxia in TrkAIII mRNA splicing was supported by significant elevation of HIF2a mRNA expression in invasive PIT1 PitNETs. These data demonstrate that alternative TrkAIII splicing, potentially promoted by hypoxia, occurs frequently in PitNETs and may associate with IF consistent with intracellular TrkAIII activation, supporting a potential role for TrkAIII in PitNET pathogenesis and progression, identifying TrkAIII as a novel potential target in refractory PitNETs.