Puma is an essential mediator of p53-dependent and –independent apoptotic pathways

1. Puma is an essential mediator of p53-dependent and –independent apoptotic pathways Jeffers JR et al. Megan Kowalski & Lauren Chmielewski November 1, 2005

2. p53 • Is a transcription factor, tumor suppressor • Sensor of DNA damage, hypoxia, oncogene activation • Activation results in cell cycle arrest or apoptosis • p53’s apoptotic functions are required for its tumor suppressor activity • Targets include: • p21Cip (a cyclin D,E-dependent kinase inhib) • Pro-apoptotic proteins • Mdm2 (negative regulator of p53)

3. Regulation of p53 Sherr CJ et al. Cancer cell Aug (2002) 2; 103-112

4. p53 – Necessary for Apoptosis? ? Cdk inhibitors Pro-apoptotic proteins Yu J, Zhang L. Cancer Cell: Oct 2003, pp 248-9.

5. 2 Mechanisms of Apoptosis via mitochondria via death receptor ligation Zimmerman KC et al.Pharm & ther 92 (2001) 57-70.

6. Apoptosis • http://www.ncbi.nlm.nih.gov/books/bookres.fcgi/mcb/ch23anim2.mov Lodish H et al. Mol Cell Bio 4th Ed online. Ch 23.8

7. Apoptotic Phenotype Apoptotic changes. T-cells undergoing apoptosis in vitro and in the thymus after activation. A: Blebbing (left) and nuclear condensation (right). Lodish H et al. Mol Cell Bio 4th Ed online. Ch 23.8 Zimmerman KC et al.Pharm & ther 92 (2001) 57-70.

8. Why Do We Care?p53 and Cancer • p53 pathway disrupted > 50% of human cancers • Finding p53’s target for mediating apoptosis could be exploited for either • sensitizing cancer cells resistant to therapies • conferring resistance on WT cells during therapy • So how does p53 mediate apoptosis?

9. Puma • Puma/Bbc3 = p53 upregulated moderator of apoptosis/Bcl2 binding component-3 • is a BH3-only pro-apoptotic Bcl2 family member • Expression induced by • p53 • Glucocorticoids (corticosteroids involved in metabolism and have anti-inflammatory properties. Examples include prednisone and cortisol.) • Serum deprivation

10. Bcl2 Family Proteins Puma Zimmerman KC et al.Pharm & ther 92 (2001) 57-70.

11. Previous Findings • Activated Puma associates with mito and induces apoptosis when overexpressed in vitro. • Puma’s apoptotic activity requires its BH3 domain • Puma knockouts in colorectal cancer cells showed that Puma was required for apoptosis induced by p53, hypoxia, and DNA-damaging agents in vitro. • But no studies done in intact animal…

12. Generation of Puma-/- MiceFigure 1 Deleted exons 1-3, includes transcription & translation start sites and BH3 domain

13. Puma Knockouts Lack WT Allele - Figure 1 Heterozygous animals identified using PCR & Southern Blot analysis Heterozygotes back-crossed to generate homozygous KOs PCR & Western used to show Puma not expressed in hematopoietic cells of KO mice

14. Figure 1 Summary • Puma-/- mouse generated successfully • KO mice appeared phenotypically normal • Since model is viable, Puma not an essential protein • Beware germline KO: compensatory mechanisms may taint results

15. Role of Puma in Thymocyte Apoptosis? • Normally, thymocytes sensitive to γ-IR-induced DNA damage • γ-IR-induced apoptosis in thymocytes requires p53 • However, p53-deficient thymocytes undergo apoptosis in response to glucocorticoids • Both a p53-dependent and p53-independent pathway for apoptosis exists in thymocytes • Used IR (DNA damage) and dexamethasone (a glucocorticoid) to assess role of Puma in apoptotic response in thymocytes

16. Role of Puma in Thymocyte Apoptosis – Figure 2 Exposed thymocytes from the above mice to dexamethasone (Dex), IR, or just vehicle and then assessed their viability by using propidium iodide staining and FACS.

17. Methods: PI Staining Propidium Iodide intercalates with dsDNA or RNA. The stain is impermeable to the membrane of viable cells and is excluded. PI can only penetrate cells with compromised membranes (dead/dying cells). PI is excited by the argon-ion laser and is therefore useful for detecting and sorting dead cells by flow cytometry. Above: PI used to stain nuclei red. www.utoronto.ca/ krause/images/Figure2.jpg

18. Fluorescent-Activated Cell Sorter (FACS) • Cells are tagged with fluorescent labels • Cell passing through laser beam is monitored for fluorescence • Droplets containing single cells are given – or + charges depending on whether cell is fluorescent or not • Droplets are deflected by an electric field into collection tubes according to charge • Cell concentration must be adjusted so that most droplets contain no cells and flow to a waste container with any cell clumps • Read collected cells using FACS software ©2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter

19. Puma-/- Thymocytes are Resistant to IR-Induced Apoptosis. Fig 2. Red= Viable Blue= Dead In absence of stress, all strains were viable In response to IR, Puma-/- maintained viability despite presence of p53 Puma-/- cells refractory to Dex-induced apoptosis. Additional proteins involved? Bim?

20. Bim • Another BH3-only pro-apoptotic protein • Expression is induced by glucocorticoids • Like Puma KO, Bim KO cells refractory to Dex-induced apoptosis • Bim-/- no effect on survival of thymocytes after IR (does not mediate IR-induced cell death)

21. Bax-Deficient Thymocytes Figure 2 • Bax is another pro-apoptotic Bcl-2 family protein • Previous studies showed requirement of Bax in Puma-mediated apoptosis • Like Bim, Bax not required for IR-induced p53/Puma-mediated cell death Whereas glucocorticoid-induced apoptosis may involve redundancy, IR-induced death does not share the same pathway

22. Figure 2 Summary • Puma is an essential mediator of p53-induced apoptosis in response to DNA damage in primary thymocytes • Puma also contributes to glucocorticoid-mediated cell death

23. No Puma, No Death (in Primary Myeloid Progenitors) Despite dual-death signals, Puma-/- myeloid progenitor cells didn’t die

24. No Puma, No Death (in Primary B Cells) Red= Viable Blue= Dead

25. Role of Puma in Thymus and CNS What is the role of Puma in DNA damage-induced death in the thymus and the developing nervous system? • P5 WT, p53-/-, Puma +/-, and Puma-deficient littermates exposed to у-IR and allowed to recover for 6 hrs • Tissues collected and analyzed for cell death by morphological and TUNEL-based assays

26. Figure 3- Thymus and у-IR Induced DNA Damage Basal levels of apoptosis in unirradiated p53-/- and WT thymi • Abundant apoptosis in WT thymus after у-IR • Apoptosis was compromised for Puma+/- mice • Almost complete resistance to у-IR-induced apoptosis for p53-/- and Puma-/- mice in thymus Puma mediates p53-dependent death in the thymus following у-IR.

27. Figure 3- CNS and у-IR Induced DNA Damage • Known that exposure of developing nervous system to IR leads to p53- dependent apoptosis • Apoptosis detected by neutral red staining and TUNEL • Widespread IR-induced apoptosis was expected and obvious in the EGL, DG, hippocampus, and SVZ of the lateral ventricle of WT mice *Arrows indicate pyknotic cells, in which the contraction of nuclear contents to a deep staining irregular mass is a sign of cell death External Granule Layer (EGL) Dentate Gyrus (DG) Hippocampus Subventricular Zone (SVZ)

28. Figure 3- CNS and у-IR Induced DNA Damage • Puma deficiency completely blocked cell death throughout the developing nervous system following у-IR treatment • Highly resistant to apoptosis as judged by the absence of pyknotic cells • TUNEL analysis confirmed virtually no TUNEL-positive cells in the у-IR treated Puma-/- CNS Puma is essential for apoptosis in thymus and developing postnatal CNS after у-IR and Puma loss recapitulates the radiation resistance observed in p53 KO mice. External Granule Layer (EGL) Dentate Gyrus (DG) Hippocampus Subventricular Zone (SVZ)

29. Puma and Oncogenes How does Puma contribute to oncogene-induced apoptosis? • Inappropriate cell proliferation stimulated by oncogenes triggers p53-dependent apoptosis of hematopoietic cells • The cell death pathway provides a mechanism for eliminating potential cancer cells and suppressing tumor development • Researchers activated c-Myc fused to the 4-hydroxytamoxifen (4-HT)-responsive domain of the estrogen receptor (ER) in primary myeloid progenitors • Progenitors were engineered to express a puromycin-resistance marker alone (Puro) or Puro and the conditional Myc-ER transgene via transduction with the MSCV-IRES-GFP control virus or the MSCV-Myc-ER-IRES-GFP virus, respectively

30. Figure 4A- Western Blot Analysis Lanes 1, 3, 5: Puro only • Expression of Myc-Err was equivalent in WT, Puma +/-, and Puma -/- cells • Induction of p21Cip1, a protein transcribed upon the activation of p53 that prevents entry into S phase, is also equivalent following 4-HT treatment

31. Figure 4A- Viability and Myc-ER expression • Viability was determined after activation of Myc-ER with 4-HT at the indicated times by trypan blue dye exclusion • Activation of Myc-ER in WT progenitors induced massive cell death • Puma -/- cells highly resistant to apoptosis (more than 50% remain viable) • Puma null cells are comparable to p53-/- myeloid cells in their response to Myc-induced apoptosis • Puma also functions as an essential apoptotic regulator that protects against hyperproliferative signals emanating from oncogenes, such as c-Myc.

32. Supplemental Figure S5A- Puma and Murine Embryo Fibroblasts • WT and p53-/- MEFs with Myc-ER transgene were treated with 4-HT and analyzed for expression of p53, Puma, and Myc-ER • No visible p53 or Puma protein for p53-/- MEFs after 24 hrs

33. Supplemental Figure S5B- MEF Viability and c-Myc • WT, p53-/-, Puma+/-, & Puma-/- MEFs were transduced with Puro-only or Puro-Myc-ER retroviruses • After Myc activation and DNA damage, cell viability assessed by trypan blue exclusion • Puma-/- MEFs were significantly more resistant to Myc-induced apoptosis as compared to WT cells • Puma-/- MEFs, like p53-/- cells, were resistant to c-Myc-induced apoptosis Puma is required for c-Myc induced death of primary MEFs.

34. Cytokines and Withdrawal • Cytokine- an extracellular signal protein or peptide that acts as a local mediator in cell-cell communication • IL-6, IL-3, and stem cell factor (SCF) required for growth and survival of bone marrow-derived myeloid progenitors • Removal of these cytokines leads to G1 cycle arrest and p53 independent apoptosis as well as reduced gene expression (Bim is an exception) • Studies have shown that removal of IL-3 suppresses Puma mRNA and expression levels

35. Figure 4B- Puma and Cytokine Withdrawal What is Puma’s role in cytokine withdrawal-induced death? • Primary BM obtained from femurs of WT, Puma+/-, & Puma-/- mice and cultured in medium with IL-3, IL-6, and SCF to promote growth • WT progenitors progressively lost viability when deprived • P53-/- cells modestly resistant to cytokine deprivation • Bim-/- cells demonstrated extended survival in media lacking cytokines, but ultimately underwent cell death • 90% of Puma-/- cells survived for 3 days in absence of cytokines and after 1 week, approx. 50% remained viable (later arrested in G1) Puma plays an important role in the intrinsic cell death response suppressed by survival factors, unlike p53.

36. Supplemental Figure S6A- Puma and Hematopoietic Development What is the role, if any, of Puma in hematopoietic development? • No significant changes in percentages of hematopoietic cell types present in WT, Puma-/- and p53 KO tissues • Normal percentages of thymic CD4- and CD8-positive cells • Thymic cellularity normal in Puma-/- mice • Aged Puma-/- mice (8 months) also failed to reveal any hematopoietic defects

37. Supplemental Figure S6B- Puma and Hematopoietic Development • Normal percentages of Thy1.2- and B220-positive cells, as well as Gr-1- and Mac-1 positive cells in the bone marrow of WT, Puma-/-, and p53-/- mice • Additional markers and stains analyzed in other tissues Normal hematopoiesis occurs in Puma-/- mice. Like p53, Puma may contribute to cell death within these cell types only during pathological stress responses. WT Puma-/- p53-/-

38. Puma and BAX • Homozygous deletion of Puma in colon carcinoma HCT116 cells that were previously targeted not to express p21Cip1 rendered these double-KO cancer cells resistant to p53-mediated apoptosis • Previously experiments found that Bax was required for Puma-mediated cell death in colon carcinoma cells • Puma is required for γ-IR induced death of thymocytes and Bax is dispensable for this response • Puma is essential for γ-IR induced apoptosis throughout the developing CNS, including the retina, which does not require Bax Puma is required for apoptosis in cell types and under stress conditions where Bax is dispensable.

39. Primary Conclusions • Puma is a critical mediator of p53 induced cell death & accounts for nearly all apoptotic activity attributed to p53 in primary hematopoietic & CNS cells in response to γ-IR or oncogenes (cMyc) • Endogenous WT p53 is not sufficient in the absence of Puma to induce efficient cell death • Puma is required for p53-mediated cell death which most like occurs through the direct transcriptional induction of Puma expression by p53 & the subsequent association of Puma with Bcl-2 family members at the mitochondria • Remarkably, the loss of just Puma has a profound effect on apoptosis after DNA damage and other apoptotic stimuli • Puma is essential to the induction of apoptosis during DNA damage

40. Secondary Conclusions • Recent studies indicate that p53’s apoptotic activity is the primary tumor suppressor function because tumors have been shown to tolerate wt p53 if appropriate survival methods are implemented. • Results of this paper suggest that the combined activities of p53 cell cycle arrest and cell death serve as checkpoints in cancer • Consistent with this hypothesis, Puma KO mice (> 6 months) do not appear to be inherently predisposed to developing spontaneous tumors • These mice lack the ability for p53-mediated apoptosis, but they do have wild type p53 for inducing cell cycle arrest if necessary • Could other proteins (when no Puma) be involved/compensating in p53-mediated apoptosis?

41. Future Directions • Does p53 directly activate PUMA transcription to initiate apoptosis in vivo or via transcription independent mechanisms? • Is Puma related to other apoptotic mediators of p53? • Does Puma deficiency mimic p53 deficiency in its ability to rescue the developmental defects caused by MDM2 knockout? • Why do some cells undergo cell cycle arrest rather than apoptosis in response to p53, even though Puma is induced? • Can Puma sensitize p53 mutant cells to anticancer drugs? • Determine whether spontaneous tumor incidence increases in aged Puma deficient mice or in cooperation with other genetically predisposed alterations.