Cell Cycle I Molecular Cell Biology November 6, 2014

1. Cell Cycle IMolecular Cell BiologyNovember 6, 2014 Stephen Oh, M.D., Ph.D. Assistant Professor Division of Hematology

2. Outline • Overview of the cell cycle • Cell cycle regulation – fundamental concepts • Cancer as a fundamental disruption in cell cycle regulation

3. What is the basic function of the cell cycle? • Accurately duplicate the vast amount of DNA in chromosomes • Segregate the copies precisely into genetically identical daughter cells Figure 17-2 Molecular Biology of the Cell, 4th Edition

4. The phases of the cell cycle • G1 – gap between M and S phases • S – DNA replication • G2 – gap between S and M phases • M - mitosis • Interphase ~23 hours • M phase ~1 hour Figure 17-3. Molecular Biology of the Cell, 4th Edition Why are gap phases needed?

5. What critical features are needed for proper guidance through the cell cycle? Figure 17-13 Molecular Biology of the Cell, 4th Edition

6. What critical features are needed for proper guidance through the cell cycle? • A clock, or timer, that turns on each event at a specific time • A mechanism for initiating events in the correct order • A mechanism to ensure that each event is triggered only once per cycle • Binary (on/off) switches that trigger events in a complete, irreversible fashion • Backup mechanisms to ensure that the cycle can work properly even when parts of the system malfunction • Adaptabilityso that the system's behavior can be modified to suit specific cell types or environmental conditions Figure 17-13 Molecular Biology of the Cell, 4th Edition

7. The cell cycle is primarily regulated by cyclically activated protein kinases Figure 17-15, 17-16 Molecular Biology of the Cell, 4th Edition

8. Evolution of cell cycle control: from yeast to humans Malumbres M, Nature Reviews Cancer 2009

9. Overview of major cyclins and Cdks of vertebrates and yeast Table 17-1. Molecular Biology of the Cell, 4th Edition

10. Overview of major cyclins and Cdks of vertebrates and yeast Bardin AJ, Nature Rev Mol Cell Biol 2001

11. Cdk activity is regulated by inhibitory phosphorylation and inhibitory proteins Why is cell cycle progression governed primarily by inhibitory regulation? Figure 17-18, 17-19. Molecular Biology of the Cell, 4th Edition

12. Cell cycle control depends on cyclical proteolysis Figure 17-20. Molecular Biology of the Cell, 4th Edition

13. Mechanisms controlling S-phase initiation Figure 17-30. Molecular Biology of the Cell, 4th Edition

14. DNA damage leads to cell cycle arrest in G1 Figure 17-33. Molecular Biology of the Cell, 4th Edition

15. Overview of the cell cycle control system Figure 17-34. Molecular Biology of the Cell, 4th Edition

16. Summary of major cell cycle regulatory proteins Table 17-2. Molecular Biology of the Cell, 4th Edition

17. Mitogens stimulate cell division Figure 17-41. Molecular Biology of the Cell, 4th Edition

18. Excessive stimulation of mitogenic pathways can lead to cell cycle arrest or cell death Figure 17-42. Molecular Biology of the Cell, 4th Edition

19. Extracellular Growth Factors Stimulate Cell Growth Figure 17-44. Molecular Biology of the Cell, 4th Edition

20. Extracellular Survival Factors Suppress Apoptosis Figure 17-47. Molecular Biology of the Cell, 4th Edition

21. Intracellular signaling networks related to cell proliferation and cancer Hanahan and Weinberg, Cell 2011

22. Myeloproliferative neoplasms are clonal disorders derived from hematopoietic stem/progenitor cells Primary myelofibrosis JAK2 V617F Essential thrombocythemia Polycythemia vera

23. JAK-STAT activation is a hallmark of myeloproliferative neoplasms TPO G-CSF JAK2 V617F P P JAK2 JAK2 P P STAT3/5 STAT3/5 P P P P STAT3/5 STAT3/5 Proliferation/Survival STAT3/5 STAT3/5

24. Dysregulated signaling networks in myeloproliferative neoplasms TPO G-CSF SCF FLT-3L TLRs Ifna LNK LNK SOCS JAK2 V617F Rux P P P P P P JAK1 JAK1 JAK2 JAK2 P TBK1 P P P P PI3K STAT3/5 STAT1 STAT1 IKKε STAT3/5 RAS IKKγ AKT P RAF CBL IKKβ IKKα P P P P P P P P P P S6K MEK P ERK IkBα P NFkB S6 IkBα NFkB NFkB CREB P STAT1 STAT3/5 STAT3/5 STAT3/5 PIM1 BAD IkBdegradation Proliferation/Survival TNFα PIM1 STAT3/5 STAT3/5 Proliferation/Survival TNFα, GM-CSF IkBα P Cell cycle inhibition/Apoptosis STAT1 P P P STAT1 STAT1 STAT1

25. Spectral limitations of flow cytometry can be overcome with elemental mass cytometry >30 parameters with single cell resolution Metal conjugated antibodies Mass channel readout CyTOF2 mass cytometer Labeled cells

26. How can we visualize data in 30+ dimensions? Bendallet al Science 2011 SPADE links related cell types in a multidimensional continuum of marker expression

27. SPADE identifies relevant cell subsets including HSPC HSPC Low High CD34 median expression:

28. Cell cycle analysis via mass cytometry Behbehani et al, Cytometry 2012

29. Cell cycle analysis via mass cytometry Behbehani et al, Cytometry 2012

30. Cell cycle regulators are frequently disrupted in cancer Malumbres M, Nature Reviews Cancer 2001

31. Overview of CDK inhibitors in clinical development for cancer therapy Results thus far have been somewhat disappointing – why? Malumbres M, Nature Reviews Cancer 2009

32. Suggested reading • Alberts et al., Molecular Biology of the Cell, 4th Edition, Garland. Updated 2001. Chapter 17. • http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.TOC&depth=2 • Malumres M, Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer. 2009 Mar;9(3):153-66. • http://www.nature.com/nrc/journal/v9/n3/full/nrc2602.html • Hanahan and Weinberg. Hallmarks of Cancer: The Next Generation. Cell. 2011 Mar 4;144(5):646-74. • http://www.sciencedirect.com/science/article/pii/S0092867411001279 • Anand S, Huntly BJ. Disordered signaling in myeloproliferative neoplasms. HematolOncolClin North Am. 2012 Oct;26(5):1017-35. • http://www.sciencedirect.com/science/article/pii/S0889858812001281 Contact: stoh@dom.wustl.edu