Abstract Neuroblastoma is a common malignant tumor in children, arising in the sympathetic nervous system. The tumor cell differentiation state influences clinical outcome, and induction of differentiation has been considered as a promising therapeutic strategy for neuroblastoma. Retinoid acid (RA) is an effective inducer of neuroblastoma cell differentiation and has been used in clinic for treatment of high-risk neuroblastomas. However, resistance to RA presents a major obstacle to successful RA-based therapy. Therefore, identification of target genes that mediate the anti-neoplastic activity of RA is of paramout importance, as targeting these genes offers opportunities to bypass resistance to RA. A variety of experimental evidence has linked RA action to the activation of HOX genes, which function as master regulators of development and cell fate specification. Particularly, it has been shown that RA induces several HOXD genes in neuroblastoma cells including HOXD1, D8 and D9. Our microarray analysis confirmed the predominant upregulation of HOXD genes in the human neuroblastoma cell line BE(2)-C cells following RA treatment. To investigate the functional significance of the RA-induced upregulation of HOXD genes, we conducted a comprehensive functional analysis of HOXD genes in regulation of neuroblastoma cell proliferation and differentiation. The HOXD cluster contains 9 genes (HOXD1, D3, D4, and D9-13) that are positioned from 3’ to 5’ within the cluster, with HOXD1 at the 3’ end and HOXD13 the 5’ end. We established BE(2)-C-derived cell lines with inducible expression of individual HOXD genes. Induction of HOXD1, D12 or D13 had no significant effect on BE(2)-C cell growth and differentiation. By contrast, individual induction of other HOXD genes caused G1 arrest, which was associated with downregulation of cyclins and CDKs required for cell cycle progression through the S, G2 and M phases. Ectopic expression of HOXD3, D4, D8, D9, D10 or D11 also induced morphologic changes characteristic of neuronal differentiation. Interestingly, HOXD8 and D9 appeared to be most potent in the induction of G1 arrest and differentiation. These findings suggest that HOXD genes positioned in the middle of the cluster have higher differentiation-inducing activity compared to those positioned more 5’ and 3’ in the cluster. Thus, pharmacological activation of HOXD8 and HOXD9 may represent a therapeutic strategy for RA-resistant neuroblastomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 243. doi:10.1158/1538-7445.AM2011-243