Moreover, C. elegans is an appropriate model to assess functions of VPAregulated genes; VPA induces similar responses in C. elegans as in mammalian cells, including activation of DNA damage response and developmental arrest. We hypothesized that use of in vivo models for functional validation would facilitate the translation of complex datasets into clinically useful biomarkers and molecular targets for enhancement of VPA-therapy in AML at low cost. A pre-existing human gene expression dataset of VPA resistance was complemented with an in vivo rat leukemia phosphoproteomic screen, and synthetic lethality in C. elegans was exploited as a functional validation tool. Using this strategy we identified novel conserved sensitizers and synthetic lethal interactors of VPA, as well as conserved resistance pathways converging on HSP90AB1, HSP90AA2, and MAPKAPK2. These observations, together with a functional relationship between protein acetylation and protein methylation involving UTX suggested multiple molecular mechanisms for effective anti-cancer valproic acid therapy. Spleens were excised, segmented and diluted with 0.9% NaCl. The filtered solution ) was homogenized prior to isolation of leukocytes by density gradient separation by Lymphoprep as described by the manufacturer. Phosphorylated proteins from 20 million BNML blasts were harvested using the PhosphoProtein Purification Kit according to the manufacturer��s description. Phosphorylated proteins were recovered after immobilized metalaffinity chromatography. Although VPA is a HDACi we found a striking underrepresentation of genes involved in chromatin remodeling in the above analyses, with SET and NUCB2 being the only DNA binding proteins identified in the phosphoproteomic screen. To identify functional interactions between VPA and genes participating in chromatin associated processes, we screened a focused C. elegans RNAi library that identified 43 genes that modulated VPA-induced developmental arrest of which an additional 28 synthetic lethal clones were identified, 6 of which are predicted, or known, transcriptional regulators. Although there was no direct PD325901 MEK inhibitor overlap between datasets harvested through the different methods used, the individual datasets indicated modulation of similar pathways or biological processes. To extract the common R428 Axl inhibitor processes reflected in all approaches we analyzed the overlap based on gene ontology annotation. The biological processes emerging from the three lists show remarkable similarities. In particular, TGFb and oxidative stress/MAPK signaling, ubiquitin dependent protein degradation, as well as maintenance of chromatin structure and the cytokinesis checkpoint are conserved processes modulated by VPA. Several of these pathways have been found to be regulated by VPA. The combination of VPA and the proteasome inhibitor bortezomib synergistically increased apoptosis and decreased proliferation in the AML cell line HL60. Further, genes active in the MAPK, ubiquitin-mediated proteolysis and TGFb signaling pathways have been found to be up-regulated in response to treatment with VPA and hydralazine in breast cancer patients. Next, we supplemented the primary data with predicted protein-interaction partners extracted using FunCoup to see whether a direct overlap between all methods and model systems could be revealed. As anticipated an overlap emerged for proteins extracted from all models. In order to identify genes and proteins that mediate resistance to the HDACi VPA in AML patients, we used a novel combination of models and technology that allowed us to address the mechanism of VPA induced toxicity at multiple levels.