2.  Dr. Steven Grant  Dr. Roberto Rosato  Jorge Almenara  Stefanie Coe  Dr. Allison Johnson, HHMI Coordinator

3. APOPTOSIS : Programmed cell death HDAC : Histone deacetylases are a class of enzymes HDACI : HDAC Inhibitors are involved in anticancer activity LAQ824 : HDAC Inhibitor FLUDARABINE :A nucleoside analog U937 CELLS : Leukemia cell lines

4. A class of enzymes that remove acetyl groups from A class of enzymes that remove acetyl groups from lysine amino acid on a histone. lysine amino acid on a histone. Altered expression and mutations of genes encodingAltered expression and mutations of genes encoding HDACs have been linked to tumor development HDACs have been linked to tumor development

5. Inhibits cell growth in a dose- and time–dependent manner Inhibits cell growth in a dose- and time–dependent manner Induces upregulation of p21, caspase cascade activation and Poly (ADP) ribose polymerase (PARP) cleavage Induces upregulation of p21, caspase cascade activation and Poly (ADP) ribose polymerase (PARP) cleavage

6. Inhibits DNA synthesisInhibits DNA synthesisDNA synthesisDNA synthesis Active against both dividing and resting cells.Active against both dividing and resting cells. One of the newer chemotherapy drugs which has been around and in use for a few years.One of the newer chemotherapy drugs which has been around and in use for a few years.

7.  Dose response of LAQ824 and Fludarabine to U937 cancer cells  Study of Proteins in U937 cells treated with LAQ824 and Fludarabine

9. Treatments : - Control - Control - LAQ824 (40 nM) - LAQ824 (40 nM) - FD 0.4, FD 0.5, FD 0.6, FD 0.7 (µM) - FD 0.4, FD 0.5, FD 0.6, FD 0.7 (µM) - L/F 0.4, L/F 0.5, L/F 0.6, L/F 0.7 - L/F 0.4, L/F 0.5, L/F 0.6, L/F 0.7 All LAQ824 treatments for 48 hours All LAQ824 treatments for 48 hours All Fludarabine (FD) treatments for 24 hours All Fludarabine (FD) treatments for 24 hours

10. Cells and Cell Culture Cells and Cell Culture Drugs and Chemicals Drugs and Chemicals Flow Cytometry Flow Cytometry

13. (p21, PARP, Caspase-3, Caspase-7 and Caspase-9)

14. Treatments Treatments - Control - Control - LAQ824 40 nM - LAQ824 40 nM - FD 0.4 µM - FD 0.4 µM - L/F 0.4 - L/F 0.4

15. Cells and Cell Culture Cells and Cell Culture Drugs and Chemicals Drugs and Chemicals Flow Cytometry Flow Cytometry Western Blot Analysis Western Blot Analysis

16. p21 Control LAQ824 FD 0.4 L/F 0.4 PARP

17. C L40 4h 8h 16h 24h 28h 32h 40h 48h C F0.4 4h 8h 16h 24h L/F 4h 8h 16h 24h Cleavage Fragment

18. C F0.4 4h 8h 16h 24h L/F 4h 8h 16h 24h C L40 4h 8h 16h 24h 28h 32h 40h 48h

19. C F0.4 4h 8h 16h 24h L/F 4h 8h 16h 24h Cleavage Fragment

20. LAQ824 and Fludarabine interact synergistically in U937 cancer cells inducing cell death by apoptotic events.