Oral RKS262 reduces tumor burden in a neuroblastoma xenograft animal model and mediates cytotoxicity through SAPK/JNK and ROS activation in vitro

Patients diagnosed with high-risk neuroblastoma (NB), an extracranial solid tumor in children, have metastases and low survival (30%) despite aggressive multi-modal therapy. Therefore new therapies are urgently needed. We show significant in vitro and in vivo antitumor efficacy of RKS262 in NB. RKS262 showed superior cytotoxicity (IC₅₀ = 6-25 μM) against six representative NB cell lines compared to its parent analog nifurtimox (currently in phase 2). Pre-formulated RKS262 (150 mg/kg/daily) pellets administered orally, suppressed tumor growth (60%, p = 0.021) in NB xenograft mice within 28 days. RKS262-treated SMSKCNR cells showed TUNEL-positive DNA nicks and activation of ROS, MAP Ks (SAPK/JNK), caspase-3 and p53, along with suppression of the IGF-1R/PI3K/PKC pathway and the Bcl2 family of proteins. RKS262 caused G₂/M-phase arrest and suppressed cdc-2, cyclin B1, p21 and cyclin D1/D4 expression. N-acetyl-cysteine (NAC; 10 mM) pre-treatment rescued cell viability of RKS262 (23 μM)-treated SMSKCNR cells and pre-treatment with ascorbic acid (100 μM) and a MAP K inhibitor SB203580 (20 μM) reversed SAP K/JNK, caspase-3 activation, PA RP-1 cleavage and suppression of IGF-1R, PI3K and PKC phosphorylation. Further, treatment with exogenous BDNF (50 nM) did not suppress SAP K/JNK or ROS activation due to RKS262. Rather, BDNF (50 nM), EGF (100 nM) and IGF-1 (100 nM) co-treatment with RKS262 induced a remarkable S-phase arrest rather than a G₂/M phase arrest when RKS262 was used alone. In summary, RKS262 shows oral efficacy in NB xenograft animals and induces apoptosis in vitro in SMSKCNR cells via cell cycle arrest, MAP K and ROS activation, and suppression of IGF-1R/PI3K/PKC and Bcl2 family proteins in a growth factor (BDNF/EGF/IGF-1)-independent fashion.