Abstract Developmental apoptosis of neural crest precursors is crucial in determining the final number of terminally differentiated cells such as sympathetic neurons. During neural development, cells undergo apoptosis as NGF becomes limiting. Aberrant developmental apoptosis is implicated in pediatric sympathetic nervous system tumors. When NGF becomes limiting, a developmental apoptotic pathway is activated which requires KIF1Bbeta. KIF1Bbeta is necessary and sufficient for apoptosis during NGF withdrawal. KIF1Bbeta maps to 1p36.2, a frequently deleted region in neuroblastomas, and a neural crest-derived cancer. We identified that KIF1Bbeta-induced apoptosis requires RNA/DNA helicase DHX9. KIF1Bbeta interacts with DHX9 to enhance translocation and accumulation of cytoplasmic DHX9 in the nucleus, resulting in transcription of apoptotic XAF1. Transcription-incompetent DHX9 is unable to potentiate KIF1Bbeta-induced cell death. Knockdown of DHX9 also protects from KIF1Bbeta-induced cell death whereas KIF1Bbeta loss-of-function domains or patient-associated point mutants are unable to translocate and accumulate cytoplasmic DHX9 in the nucleus, impairing XAF1 expression. Furthermore, XAF1 silencing protects from KIF1Bbeta-induced and NGF withdrawal-mediated apoptosis in vitro as well as promotes tumor growth in vivo whereas XAF1 overexpression is necessary and sufficient to induce apoptosis in vitro and delays tumor growth in vivo. Conditional knockout of KIF1Bbeta in the superior cervical ganglia neurons of mouse pups also specifically ablates XAF1 expression in vivo and ex vivo, suggesting that KIF1Bbeta and XAF1 are tightly regulated and act along the same pathway. Clinically, tissue microarray analysis of pre-treatment or post-treatment neuroblastoma patients who are 1p-intact or 1p-deleted, revealed strong prognostic significance of XAF1 expression in disease stratification. Our findings provide a mechanistic understanding on the development of neuroblastoma through unexpected candidates of tumorigenesis. Citation Format: Zhang'e Choo, Rachel Yu Lin Koh, Karin Wallis, Timothy Jia Wei Koh, Chik Hong Kuick, Veronica Sobrado, Amos Hong Pheng Loh, Shui Yen Soh, Susanne Schlisio, Kenneth Tou En Chang, Zhi Xiong Chen. Unusual suspects identified in neuroblastoma: An unexpected tumor suppression pathway. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A47.