DIPG-04. COMBINED TARGETING OF CALCIUM SIGNALLING AND RTK/PI3K PATHWAY IS A NOVEL THERAPEUTIC APPROACH AGAINST DIFFUSE INTRINSIC PONTINE GLIOMA

摘要

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive paediatric brainstem tumour, with a peak incidence in middle childhood and a median survival of less than 1 year in the majority of cases. The dismal prognosis associated with DIPG is exacerbated by the repeated failure of over 250 clinical trials to improve survival over standard radiotherapy. We performed a high throughput screen (HTS) of 3500 clinically available compounds, which identified 33 active agents against DIPG. Furthermore we performed a combination HTS based on these 33 agents together with 50 chemotherapeutic agents against each other, resulting in 3,403 combinations. This unbiased combination HTS identified >40 active combinations, with Calmidazolium / Nilotinib and Calmidazolium / Temsirolimus showing the most potent synergy. These promising candidates were selected for further investigation in vitro using cytotoxicity assays, colony formation assays, flow cytometry assessing apoptotic events and western blot to elucidate mechanisms of actions. The two calmidazolium-based combinations demonstrated a synergistic reduction in DIPG cell viability at therapeutically relevant concentrations. Further, calmidazolium combinations significantly enhanced the anti-proliferative effect of radiation when administered together and assessed by colony formation assays. The induction of apoptosis was successfully demonstrated in a time-dependent manner and incidentally corresponded to a reduction in CD133+ expressing DIPG cells. Interrogation of mechanisms of action, found that the combination therapies acted via synergistic inhibition of PI3K/AKT/mTOR pathway and key partners in calcium signalling such as calcineurin and oncogenic transcription factor NFAT. The PI3K/AKT/mTOR pathway has been previously shown to play a key role in tumour development in DIPG while deregulated calcium signalling has been reported in adult brain tumours. Our results suggest that the Calcium Signalling pathway is a previously unrecognised targetable network in DIPG. Furthermore combination with oncogogenic PI3K/AKT/mTOR represents a potentially novel, potent combination therapy for this devastating paediatric brainstem tumour.