Hypoxia-induced alternative TrkA splicing and reoxygenation-dependent TrkAIII activation: A cycling hypoxia-regulated resistance mechanism in neuroblastoma.

At any specific time, 20 to 30% of solid tumour microenvironments are conditioned by acute transient hypoxia followed by reoxygenation (cyclic hypoxia) caused by interruptions in blood flow within immature malformed blood vessels, which combined with chronic diffusion hypoxia, ensure that 100% of tumour cells will eventually experience hypoxia. Hypoxia followed by reoxygenation causes reperfusion injury and represents an important cytotoxic selection mechanism for resistant tumour cell subpopulations. Reperfusion injury involves the endoplasmic reticulum (ER) stress-response, ER Ca2+release and mitochondrial Ca2+ uptake, resulting in a cytotoxic metabolic burst, mitochondrial transition permeability pore opening and cell death. Neuroblastomas (NBs) are aggressive, heterogeneous, highly malignant paediatric tumours of embryonic neural crest origin that, in general, carry poor prognosis. Previously, we reported that advanced stage, relapsed and metastatic NBs associate with enhanced expression of an oncogenic alternative splice variant of the tropomyosin-related kinase A (TrkA) neurotrophin receptor, TrkAIII, providing a mechanism through which NB suppressing signals from fully spliced TrkA can be converted into oncogenic signals. The TrkA gene is comprised of 17 exons, alternative TrkA splicing is restricted to exons 1 to 8 and TrkAIII, characterized by exons 6 and 7 skipping, is the only in-frame alternative splice variant expressed by NB cells, detected to date. TrkAIII oncogenic activity, confirmed by NIH3T3 transformation and promotion of primary and metastatic tumourigenic activity in NB models, results from omission of the D4 IG-like domain, required for cell surface receptor expression and the prevention of spontaneous receptor activation. As a consequence, TrkAIII receptors exhibit intracellular retention and, compared to fully spliced TrkA receptors, more readily sustain spontaneous ligand-independent intracellular activation in NB cells, resulting in pro-survival (IP3K/Akt, Bcl-xL, Mcl1), pro-angiogenic (MMP-9, VEGF) and antioxidant (SOD2) signalling and gene expression, within a stem cell-like context. In this presentation, we will provide evidence that hypoxia (<0.1% [O2]) induces quiescence and promotes alternative TrkA mRNA splicing in NB cells resulting in TrkAIII expression but prevents TrkAIII activation, whereas TrkAIII is activated upon reoxygenation. We will demonstrate that TrkAIII activation induced by hypoxia followed by reoxygenation associates with activation of the ER stress response, involves ER Ca2+ release and enhanced intracellular oxidative conditions, and significantly mitigates the cytotoxicity of cyclic hypoxia and stress-induced Ca2+-transients. We propose that hypoxia-induced alternative TrkA splicing, TrkAIII expression and reoxygenation-dependent TrkAIII activation represent components of a novel, targetable, cyclic hypoxia-dependent mechanism for selecting quiescent resistant NB subpopulations within the tumour microenvironment. This helps to explain TrkAIII association with advanced stage, metastatic and relapsed NBs and also a recent case report of remarkable long-term (>5 years and ongoing) robust response to compassionate entrectinib Alk/Trk inhibitor therapy, within a context of exhausted therapeutic options, in a youngster with refractory, relapsed, advanced stage metastatic TrkAIII expressing NB (Treis et al., JCO Precision Oncology 2022, doi:10.1200/PO.21.00271). Finally, our observations support the therapeutic potential of targeting TrkAIII in NB, whenever detected, in order to enhance natural and/or induced reperfusion injury and drug-induced stress-mediated killing within the tumour microenvironment, by reducing stress-resistance in TrkAIII expressing NB cells.