1. Sameer Ahmad Batoo Mentor: Francisco Hernandez- Illizaliturri

2. Overview Histones are core structural components of chromatin; DNA is wound around histones, and histones further associate to become and form chromatin. Histone deacetylation inhibitors (HDAC) inhibitors induce accumulation of acetylated histones which leads to relaxation of chromatin structure and promotes access to transcriptional machinery and RNA polymerase

3. There are 2 Classes of DACs ( I and II ), Which Act on Different Target Proteins 3 HDAC4 Class II DACs act on NON-HISTONE proteins located in the cytoplasm (e.g. HDAC6) Class I DACs act on HISTONES and TRANSCRIPTION FACTORS located in the nucleus There are 2 main classes of DACs HDAC1 HDAC2 HDAC3 HDAC8 HDAC5 HDAC7 HDAC9 HDAC6 HDAC10 HDAC7

4. Genetic Variations and Epigenetic Changes Can Both Contribute to Oncogenesis 4 DNA Mutations/translocations Replication errors GENETIC Chromatin EPIGENETIC Transformed cells Open/closed chromatin Enzyme modification errors Altered DNA/mRNA/proteins DNA sequence altered Altered mRNA/proteins DNA sequence not altered Oncogenesis Can be caused by: Abnormal modifications to histone proteins Abnormal DNA methylation Can be caused by: Abnormal modifications to histone proteins Abnormal DNA methylation

5. Acetylation of Histones by HAT Allows Gene Expression 5 Acetylation by histone acetyltransferases (HATs) allows transcription and gene expression Acetylated Histone Open chromatin Transcription factors can access DNA Deacetylated Histone Closed chromatin Transcription factors cannot access DNA HAT HISTONE ACETYLATION Ac: acetyl group Transcription factors –Ac Ac–

6. Deacetylation of Histones by HDAC Can Prevent Gene Expression 6 Acetylation by histone acetyltransferases (HATs) allows transcription and gene expression Deacetylation by histone deacetylases (HDACs) can prevent transcription and gene expression HAT HISTONE ACETYLATION HISTONE DEACETYLATION HDAC Acetylated Histone Open chromatin Transcription factors can access DNA Deacetylated Histone Closed chromatin Transcription factors cannot access DNA Ac: acetyl group HDAC depicts a class I deacetylase Transcription factors –Ac Ac–

7. In Normal Cells, Balanced HAT and HDAC Activity Results in Regulated Gene Expression 7 Normal Cell DeacetylationAcetylation Histone deacetylation prevents gene expression Histone acetylation allows gene expression HDAC HAT Ac: acetyl group TF: transcription factors HDAC depicts a class I deacetylase –Ac Ac– TF

8. In Tumor Cells, Imbalanced HAT and HDAC Activity Can Result in Deregulated Gene Expression 8 Tumor Cell Unchecked Cell Growth and Survival Decreased Tumor Suppressor Gene Activity (p21, p27) Increased HDAC Activity Decreased HAT Activity HDAC HAT Ac: acetyl group TF: transcription factors HDAC depicts a class I deacetylase TF –Ac Ac–

9. HDAC Inhibition Restores Gene Expression in Tumor Cells 9 HDAC DAC Inhibition Increases Acetylation of Histones HAT DAC Inhibitor Increased Tumor Suppressor Gene Activity (p21, p27) Cell-Cycle Arrest and Differentiation Normalized Cell Ac: acetyl group TF: transcription factors HDAC depicts a class I deacetylase –Ac Ac– TF

10. Histonep53  -tubulin HSP90 HIF-1  Proteins modulated by DACs Deacetylase (DAC) Activity on Proteins is Associated with Downstream Effects that Promote Oncogenesis DAC Tumor suppressor gene activity Loss of tumor suppressor function Microtubule depolymerization/ aggresome formation VEGFOncoproteins Downstream effects Cell-cycle arrest Apoptosis Cell motility and Invasion Cell proliferation and survival Angiogenesis Tumor effects

11. DAC Histonep53  -tubulin HSP90 HIF-1  Proteins modulated by DACs Pan-DAC Inhibition Interferes with the Multiple Hallmarks of Cancer Cell-cycle arrest Apoptosis Cell motility and Invasion Cell proliferation and survival Angiogenesis Tumor effects Tumor suppressor gene activity Loss of tumor suppressor function Microtubule depolymerization/ aggresome formation VEGFOncoproteins Downstream effects DAC Inhibitor

12. Pan-DAC Inhibition May Have Potential in Several Cancers 12 Histone  -tubulin HSP90 HIF-1  DACs Hematologic & Solid Tumors Breast, Multiple Myeloma RCC, Melanoma CML, Breast, Prostate, NSCLC 50% of Cancers DAC Inhibitor p53

13. Protocol of my study MATERIALS- Cell Lines- Raji Raji 2R Raji 4R RL RL4RH

14. HDAC Inhibitors Entinostat Vorinostat LBH589 Dose- 10µm/ml

15. Anti CD20 Antibodies Rituximab Ofatumumab HA 20 Dose-1µg/ml

16. Other Materials RPMI Isotype ( herceptin) Human serum Cr51 Triton 5%

17. Method RPMI + Cells (5 million) Entinostat + Cells (5 million) Vorinostat + Cells (5 million) LBH589 + Cells (5million)

18. Continued Labelling cells with Cr51. (Incubation 2 hrs) Adding antibodies. ( 30 min for binding) Adding human serum ( Incubation 6 hrs) Adding Triton 5% to max release row of cells.

19. PlaceboEntinostatVorinostatLBH589 RPMI Ritux Ofatu HA20 Isotyp e HS Triton( 5%)

20. 4. Reading samples. 5. Calculating % of Lyses using the formula: % Lyses = [51Cr release from sample — 51Cr release from control]/ [51Cr release from maximum release — 51Cr release from control] x 100

21. Results

22. Raji

23. Raji 2R

24. Raji 4R

25. RL

26. RL 4RH

27. CD20 counting using Flow cytometer Possible Mechanism Of Decreased Cell Killing- ? Decrease In CD20 Conts.

28. RPMIEntinostatVorinostatLBH589 CD20 Isotype Raji cells

29. Results Decreased complement mediated cell killing by Rituximab and other CD20 antibodies in lymphoma cell lines (both rituximab sensitive and resistant cells) after cells were pre treated with HDACI in a dose of 10µM/ml and for a incubation period of 48 hours.

30. Conclusion Before Invalidating the use of above combination in the treatment of various lymphomas we need to do further studies like incubating cells for less or more than 48 hours; reversing the sequence of events like treating the cells with Anti-CD20 antibody prior to adding the HDACI. Perhaps increasing dosage.

31. References 1. Garber K. HDAC inhibitors overcome first hurdle. Nat Biotechnol 2007;25:17–9. 2. Kouzarides T. Histone Acetylases and Deacetylases in Cell Proliferation. Curr Opin Genet Dev 1999;9:40–8. 3. Mehnert JM, Kelly K. Histone Deacetylase Inhibitors: Biology and mechanism of Action. Cancer J 2007;13:23–9. 4. Minucci S, Pelicci PG. Histone Deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer 2006;6:38–51. 5. Bolden JE, Peart MJ, Johnstone RW. Nat Rev Drug Discov 2006;5:769–84. 6. Esteller M. Nat Rev Genet 2007;8:286–98 7. Yoo CB, Jones PA. Nat Rev Drug Discov 2006;5:37–50 8. Marks P et al. Nat Rev Cancer 2001;1:194–202.

32. Acknowledgement Dr Francisco Hernandez-IllizaliturriDr Myron Czuczman Dr Khalid J Qazi Dr Michael Krabak