Drug resistance is well known in chronic lymphocytic leukemia therapy, where multidrugresistant

lymphoid cancers defy the most powerful chemotherapeutics available. In this

respect, there is a renewed interest in multifocal anti-cancer compounds which simultaneously

inhibit multiple signal transduction/survival pathways. We have identified Withaferin A (WA)

as a very effective tumor selective anti-cancer compound, which chemosensitizes various

therapy resistant cancers, by binding molecules from different survival pathways. Hence,

there is an essential need for investigations that can 1) provide a global view of cellular

targets of WA and 2) select from this pool those interactions that are responsible for the

anticancer effect. Such studies can lead to faster optimization of WA treatment, understanding

of off-target side effects and the ability to minimize possible toxicities early on in the process.

In this proposal, WA-specific binding proteins responsible for anti-cancer effects of WA will

be determined in the JVM2 CLL cell model or primary CD19+ enriched PBMC samples from

CLL patients and healthy donors by applying affinity based SILAC and iTRAQ proteomic

methods using biotinylated WA (active compound) or biotinylated WN (inactive WA

analogue). The established WA-specific protein network will be combined with gene

expression array datasets, by applying systems biology bioinformatic approaches (IPA,

Nextbio, GO, KEGG, STRING, GProfile, Cytoscape). Key target proteins of WA, involved in

its anti-cancer effects will be validated in affinity pull down and silencing studies in JVM2

CLLs, and CD19+ enriched PBMC samples from healthy donors or CLL patients.