2. Background HDAC inhibitors alter gene expression Show potent anti-tumour activities Possible use as chemotherapeutic agents HAT HDAC

3. However... HDI Apicidin shown to induce P-glycoprotein P-gp = Integral membrane protein involved in drug efflux and potentially multidrug resistance Aim Elucidate mechanism of Apicidin induced P-gp expression

6. Apicidin induced P-gp promoter activity is SP1 dependant Mithramycin, inhibitor of SP1 binding abolished Apicidin induced expression

7. Activation P-gp reporter deletion mutants by Apicidin requires Y-box (NF-Y) and GC box (SP1) Does not require C/EBPbor AP-1

8. Mutation analysis of promoter regions Confirms importance of Y-box and GC box

9. ChIP analysis of TF binding No Change Increase Dissociation Accumulation Active transcription

10. Kinase signalling and Apicidin induction Inhibition of PI3K, but not other kinase pathways reduces P-gp promoter activity

11. Inhibition of PI3K pathway blocks Apicidin induced P-gp

12. Effect of PI3K pathway inhibition on TF binding and SP1 phosphorylation Blocking SP1 phosphorylation by PI3K pathway inhibits dissociation of HDAC1 and recruitment of C/EBPb

14. Summary... Apicidin induces PI3K pathway, PI3K results in phosphorylationof SP1 SP1 phosphorylation signals chromatin remodelling and activation of P-gp This is independent of changes in methylation

15. Further work Effect of prolonged treatment /different dose Change in Methylation status Was methylation effected by Aza treatment Range of 0.3 to 3mM

16. Further work Cell recovery after Short term treatment Long term treatment Effect of apicidin MDR sensitive cell lines/induction of MDR by Apicidin Polymophisms/Translocations? Proof of elimination of TF binding in mutants? Binding of C/EBPb/pCAFto Y-box/each other Accumulation of HDAC2 in non-responsive cell line