With or Without You: Studying the Requirement of p53 for Anti-Cancer Responses to Nuclear Export Inhibitors

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1 University of Colorado, Boulder CU Scholar Undergraduate Honors Theses Honors Program Spring 2016 With or Without You: Studying the Requirement of p53 for Anti-Cancer Responses to Nuclear Export Inhibitors Andrea E. Doak University of Colorado, Boulder, Follow this and additional works at: Part of the Cancer Biology Commons, Cell Biology Commons, Medicinal Chemistry and Pharmaceutics Commons, and the Molecular Biology Commons Recommended Citation Doak, Andrea E., "With or Without You: Studying the Requirement of p53 for Anti-Cancer Responses to Nuclear Export Inhibitors" (2016). Undergraduate Honors Theses This Thesis is brought to you for free and open access by Honors Program at CU Scholar. It has been accepted for inclusion in Undergraduate Honors Theses by an authorized administrator of CU Scholar. For more information, please contact

2 With or Without You: Studying the Requirement of p53 for Anti-Cancer Responses to Nuclear Export Inhibitors Andrea Doak Department of Molecular, Cellular, and Developmental Biology University of Colorado, Boulder Defense Date: April 4 th, 2016 Thesis committee: Thesis Advisor: Dr. James Orth, Molecular, Cellular, and Developmental Biology Committee Members: Dr. Jennifer Martin, Molecular, Cellular, and Developmental Biology Dr. Xiang Wang, Chemistry and Biochemistry

3 ABSTRACT Exportin-1 (XPO-1) is responsible for the movement of cargo proteins out of the nucleus and into the cytoplasm. Selective inhibitors of nuclear export (SINE) bind XPO-1 at cysteine-528, which results in the sequestration of cargo proteins in the nucleus. SINE drugs are currently being developed and tested in the treatment of many types of cancers. One of the cargos, p53 may play an important role in the efficacy of SINE. To test the necessity of p53 in the action of SINE drugs, matched pairs of cell lines with wildtype or functionally disrupted p53 were analyzed for differences in their cell fates after treatment. The drug Nutlin targets the p53 degradation pathway and was used in comparison to SINE. SINE-induced cell death and arrest can occur independently of p53. Nutlin induced arrest in G-1 phase independent of p53, but cell death is largely dependent on p53. The two drugs in combination result in a strong death response in cells with and without p53. BACKGROUND Cancer and heterogeneity Cancer is a heterogeneous disease. A malignant growth can begin any time that a cell has accumulated enough mutations to rid itself of cell cycle checkpoints and encourage rapid growth. Each case has a unique mix of mutations combining a loss of functional tumor suppressors with the gain or up-regulation of oncogenes. Tumor suppressors are in place to protect the cells from cancer, monitoring the DNA and activating any repairs or stress responses that are necessary, eventually sending the cell to its death if the damage is too great. Oncogenes are genes that code for proteins that typically encourage normal cell growth, but have increased in number or have mutated to constantly signal the cell to grow. Once malignancy has been reached, the cells continue to mutate, creating subpopulations within the cancer. 2

4 As a result of genetic heterogeneity, cancer cells, even within the same tissue of origin, have the potential to react in different ways to a given cancer treatment. This not only means that the same treatment may not be effective for different patients, but it may also only be effective in killing a certain percentage of the cancer cells within a given tumor. There are multiple responses or cell fates that a cancer cell can experience when exposed to a treatment. It can die, succumbing to the stress created by the drug. The cell can also enter a stage called senescence, in which it stops dividing but does not die. Some cells arrest in a certain cell cycle stage, repair damage, and resume growth. Others can continue to divide, unaffected by the drug, or the population can continue to mutate and become resistant to the drug. To maximize the efficacy of drugs, we need to be able to predict which of these fates will occur and understand how cells that continue to survive can be pushed towards death. A more targeted approach Many of the standard chemotherapy treatments are meant to bring every cell in your body near death, hoping that the cancer cells will be unable to repair themselves and die before the healthy cells. These chemotherapy agents are combined in drug cocktails to minimize the chance of the disease surviving (Mukherjee, 2010). These treatments are unpleasant for the patients and have a slew of side effects. Standard chemotherapy today often includes the drugs cisplatin and paclitaxel. Side effects include: kidney poisoning, death of cells in the ear, nerve death, blood poisoning (Yamazaki, Sekine, & Saijo, 1998) (Mezencev, 2015). Growing knowledge of cancer has allowed us to make drug therapies that target proteins that we now know are involved in cancer growth. These drugs often have far fewer side effects than the older chemotherapies. Trastuzumab was recently released for the treatment of breast cancer. It targets the protein HER2 (Human epidermal growth factor receptor 2) which is 3

5 overexpressed in about 20% of breast cancer. HER2 positive breast cancers have a higher mortality rate when treated with non-targeted treatment, but the release of trastuzumab has increased survival rates with fewer side effects (Mustacchi et al., 2015) The company Karyopharm has begun testing a class of drugs known as Selective Inhibitors of Nuclear Export or SINE in clinical trials ( ClinicalTrials.Gov ). These drugs target a protein called exportin-1 (XPO-1), also known as Crm-1 in yeast. High levels of XPO-1 in cancers have been found to correlate with a higher mortality rate, making the protein not only a good target for drugs, but also a useful tool for understanding cancer development (Parikh, Cang, Sekhri, and Liu, 2014). Xpo-1 in cancer Many researchers noticed a change in the spatial localization of several tumor suppressors and proto-oncogenes/oncogenes in cancer cells, many of which contain the nuclear export signal (NES) (Dickmanns, Monecke, & Ficner, 2015). The protein XPO-1 recognizes the NES signal on cargo proteins and binds these cargos along with RanBP and RanGTP to bring the cargo through the nuclear pore complex and into the cytoplasm, where RanGTP is hydrolyzed and XPO-1 releases the cargo. XPO-1 then returns to the nucleus where it is bound by RanGTP and is able to bind cargo again. (Dickmanns et al., 2015). 4

6 Figure 1: Proteins were found to be higher than normal in some cancer cells in the areas in which the arrow is pointing; the green changes are attributed to increased XPO-1 levels and activity, resulting in the green proteins being relocated from the nucleus to the cytosol (Dickmanns et al., 2015). Normal cells have multiple proteins in place that monitor DNA as well as multiple stimuli in the cell and can decide to repair mutated DNA or encourage the cell to commit suicide via a process called apoptosis. These proteins localize to the center of the cell in the nucleus where DNA is stored so that they are constantly in close contact with DNA (Conforti et al., 2015). Normal upkeep in a cell involves degrading old proteins and producing new ones so that the chances of damaged proteins functioning improperly are reduced. XPO-1 is responsible for binding proteins in the nucleus and shuttling them into the cytoplasm of the cell where they can be degraded. XPO-1 interacts with over 200 proteins, among them several of the important proteins that regulate the detection and repair of mutations and cell growth. Some of these are potent tumor suppressor proteins, including p53, p73, and p21, that exert dominant effects on cell 5

7 cycle progression, senescence, and apoptosis from within the nucleus (Fu & Chook.). When there is excess XPO-1 in the cell, it leads to increased degradation of cell-cycle regulatory proteins. With decreased regulation, the cancer is more likely to survive and accumulate mutations without the stress being sensed and the cells pushed to apoptosis. Figure 2: A diagram of XPO-1 activity. XPO-1 binds a protein in the nucleus (in this example p53) and exits the nucleus through a nuclear pore complex. Once in the cytosol, XPO-1 releases its cargo and then re-enters the nucleus through a nuclear pore complex, ready to bind another cargo (Karyopharm Therapuetics Inc.). Leptomycin B was the first compound identified as an XPO-1 inhibitor, originally used as an antifungal agent (Kudo et al., 1999). Leptomycin B irreversibly binds in the NES binding pocket of XPO-1 at cysteine-528, rendering it unable to bind cargo proteins for export (Dickmanns et al., 2015). When Leptomycin B made it to phase 1 clinical trials, it was found to have severe toxicity resulting in side effects such as vomiting, malaise, and profound anorexia. 6

8 The trial was immediately stopped and Leptomycin B was pulled from clinical development (Newlands, Rustin, & Brampton, 1996). SINE Selective inhibitors of Nuclear Export Karyopharm Therapeutics has developed a new class of drugs known as Selective Inhibitors of Nuclear Export (SINE). SINE drugs bind in the binding pocket of XPO-1 and prevent it from shuttling any proteins out of the nucleus, which leads to the sequestration and an accumulation of regulatory proteins in the nucleus, increasing the chances of the cell committing to apoptosis (Conforti et al., 2015), particularly if the cells are p53 and/or p73 positive, although this has not been tested directly. Early stage clinical trials suggest success with SINE drugs but it is still yet to be understood which treatment conditions and which XPO-1 proteins are most ideal or required for killing cancer cells (Walker et al., 2013). These compounds are currently undergoing several clinical trials in multiple types of cancer, including: metastatic breast cancer, Hematological cancer, Lymphoma, and Sarcoma (ClinicalTrials.Gov ). Since XPO-1 interacts with so many proteins, there are several ways that SINE drugs could work. One of the more popular hypotheses is that by blocking export of tumor suppressors such as p53, p73, and p21, SINE are able to enforce their jobs and induce cell death. Survivin is another cargo of XPO-1 that may encourage apoptosis when it is in the nucleus; it is possible that when survivin is blocked from entering the cytoplasm, death is more likely to occur. XPO-1 has also been found to interact with nucleoli, and could be limiting the cell s ability to produce proteins, eventually leading to death. These are just a few of the possible pathways for SINE drugs to work, and it could be a few or many of them combined. 7

9 Figure 3: Molecules of XPO-1 activity inhibitors. Kpt-330 is the primary drug used in this study (Turner et al., 2013) Tumor suppressors One of the most famous proteins in cancer research, p53, a transcription factor and tumor suppressor also known as the guardian of the genome, is one of the cargos of XPO-1 (Yoshimura, 2014). P53 is activated by several forms of stress and is able to affect cell fate by initiating molecular signaling pathways, depending on the state of the cell. P53 can begin a DNA damage repair pathway, arrest the cell in a certain cell-cycle stage, or send the cell into apoptosis (Kruiswijk, Labuschagne, & Vousden, 2015). Unfortunately, a large proportion of cancers lack a functional p53. In the absence of p53 there are other tumor suppressor proteins that can act as a backup, such as p73 (Schmid, Wang, and Wesierska-Gadek, 1999). It is not yet known if SINE drugs work largely due to the accumulation of p53 or if this class of drugs can work well independent of p53 and p73. SINE drugs may be able to act through p73 alone, which can replace the function of p53 in inducing cell cycle arrest, senescence, and apoptosis, particularly when p53 is lost. Identifying how SINE drugs work with and without p53 and if p73 or p21 is important to the response may help predict success or failure after treatment. 8

10 Figure 4: The various types of stress that can activate p53 (in purple) and the responses that p53 can implement on the cell. These can happen simultaneously. P53 can choose to arrest the cell and start DNA repair so that the cell does not divide with a mutated sequence of DNA (Bieging & Attardi, 2012). Nutlin The drug Nutlin activates p53 through a pathway distinct from SINE. Nutlin blocks the activity of MDM2/HDM2, an E3 ubiquitin ligase that tags p53 for degradation. MDM2 can sometimes be up-regulated in cancers, leading to lower levels of p53 and decreased chances of cell cycle arrest and cell death pathways being activated by the tumor suppressor (Momand, Wu, & Dasgupta, 2000). This drug can be used in comparison to SINE drugs and potentially in combination to increase p53 levels in the nucleus and possibly shift cell fates. If the two cause identical cell responses, then the pathways are likely to be similar as well, and p53 begins to look more responsible for the death of cells when SINE drugs are present. If the two reactions are different, then there are likely other pathways involved in the cellular response when XPO-1 is blocked, and we can begin to study the differences. P53 is not the only target of MDM2 though. MDM2 is believed to be involved in the degradation of the protein Forkhead box O 3a (FOXO3a), which is a transcription factor that can block cell proliferation, and p73, which is 9

11 related to p53 both structurally and functionally. MDM2 also interacts with histones on DNA (Marine & Lozano, 2010). So while Nutlin may give us some insight into how SINE works, it is also a complex drug that likely has several effects on the cell. These effects could overlap with those of SINE drugs, since p53, p73 and FOXO3a are also cargos of Xpo-1. FUCCI We use the fluorescent cell cycle sensor system known as FUCCI fluorescence ubiquitination cell cycle indicator to study how drugs affect the cell cycle of cancer cells in culture. The system works with an RFP tagged peptide fragment of Cdt1, which makes the nucleus red during G1 phase, and a GFP tagged peptide fragment of Geminin which makes the nucleus green during S and G2 phase and mitosis. The two probes combine near early S phase to turn the nucleus yellow. These two probes are non-functional and act only as reporters. Full length Cdt1 is recruited during G1 to the origin of replication so the cell can mark origins of DNA replication. Once replication has begun, the complex of which Cdt1 is a part of dissociates and Cdt1 is degraded. Full length Geminin blocks DNA replication at sites that have already been replicated and its levels begin to rise in S-phase and it stays in the nucleus through G2. It is believed that Geminin rises during S and G2 to prevent re-replication of the genome, so that each daughter cell will only replicate its genome once. Geminin prevents refiring at the origin of replication by binding and inactivating Cdt1 (Wohlschlegel et al., 2000). 10

12 Figure 5: FUCCI color cycling in cells in HT1080 cells. The cell is in G1 stage until 6 hours, when S phase begins and DNA replication is starting or occurring. The cell then enters G2 around 8 to 12 hours and progresses to mitosis (Marcus, Burke, DeSisto, Landesman, & Orth, 2015) We can use this fluorescent system to track how long the cell is in each cell cycle stage and where it is arresting or dying. FUCCI allows us to follow each cell individually, and characterize the different responses to SINE drugs. Figure 6: Normal cell cycle of MCF7 wild type 11

13 Figure 7: Different life cycle phenotypes observed in MCF7 cells when treated with KPT-330. Left cell attempts to divide and dies at 36 hours. Right- Cell remains in G1 for the entirety of life. GOAL The goal of this project is to characterize the impact that p53 has in cellular response to SINE drugs, using matched pair cell lines and Nutlin for comparison. Cell fate will be analyzed through multiple tests for data at the single cell and total population level. 12

14 METHODS Cell Line Culture MCF7 cell lines are grown in RPMI medium supplemented with 10% FBS and 1% Penicillin/streptomycin (Sigma). Cal51 cell lines are grown in DMEM medium supplemented with 10% FBS, 1% Sodium pyruvate (ThermoScientific), 1% Non-essential amino acids (ThermoScientific), and 1% penicillin/streptomycin/amphotericin b solution (Sigma). All cells were maintained at 37 C, with 5% CO2 and 80% humidity. Lentivirus Lentiviral vectors that encode the Cdt1 and Geminin sensors were obtained from Sakaue- Sawano (Sakaue-Sawano et al.) via material transfer agreement (James D. Orth). DNAs were prepared using Qiagen maxi-prep kit and lentiviral particles were produced using HEK293FT packaging cells. Cells were infected with virus and stable clones were obtained by dilution plating into 96 well plates and expansion Timelapse Timelapse was done following the previous protocol outlined in (Marcus et al., 2015). An Olympus IX81 inverted epifluorescence microscope was used. The microscope is equipped with a motorized X-Y-Z stage and stage-top InVivo Scientific environmental chamber that maintains the sample at 80% humidity, 37 o C, and 5% CO2. MCF7 wild type and shp53 cells were plated in 12 well glass bottom dishes (CellVis) and treated with the following conditions: Mock (untreated): DMSO at 2 µl/ml Kpt-330: 1 µm Nutlin: 10 µm 13

15 Kpt-330 at 1µM + Nultin at 10 µm Images were taken every 10 minutes using phase contrast, RFP, and GFP imaging for a total of 72 hours. Objectives used were a 20X Ph2, NA0.70 or 40X Ph2, NA0.75. Images were binned 2x2 and a 50% neutral density filter was applied to reduce required light exposure and light exposure times were kept to a minimum. Two positions were monitored in mock condition. Three positions were monitored for each of the other conditions. Cells were then tracked using the program FIJI (NIH), position and fluorescent intensities for RFP and GFP were recorded. (Exposure times here) Drug time course and immunofluorescence of fixed cells Cal51 wild type and MCF7 wild type cells were plated on glass coverslips. After hours, drug treatment was started. The same conditions that were used for the time-lapse were used on the coverslipsslips. Cal51 cells were fixed at 4, 8, and 16 hours. MCF7 cells were fixed at 4, 8, 16, and 24 hours. After treatment, cells were washed with PBS and fixed with 3.7% formaldehyde in PBS. Cells were then permeabilized with 0.2% Triton X-100. Blocking buffer was performed with 5% BSA in PBS. P53 was stained with the DO-1 monoclonal mouse antibody (Santa Cruz Biotechnology) and an Alexa-568 goat-anti-rabbit antibody (Molecular Probes). Coverslips were mounted on cleaned glass slides using Prolong Gold antifade mounting reagent (Molecular Probes). Nuclei were counterstained using DAPI (Sigma). Images were taken using a 40X Ph2 NA0.75 objective using the Olympus IX81. All images at each time point for each cell line were taken at identical exposure times so fluorescent intensity levels can be compared. 14

16 Beta-Galactosidase staining To check for senescence, cells were fixed and stained for Beta-Galactosidase. MCF7 cells were plated at on glass coverslips and after 24 hours treatment was started. Cells were then fixed and stained using the Cell Signaling Beta-Galactosidase Senescence kit. Cells were left in staining solution for 16 hours. Ideal staining time was determined using cells with no treatment and PD Slides were then mounted with Prolong Gold antifade mounting reagent (Molecular Probes). Images were taken in phase contrast with an upright Nikon E600 Widefield Microscope. Beta-galactosidase is considered a marker for senescent cells (Dimri et al., 1995). Senescent cells often show an increase in lysosome number, and Beta-Galactosidase is a lysosomal protein, typically functioning at lower ph levels between 4.0 and 4.5. When lysomsome levels are higher, there is enough Beta-Galactosidase to be detected at a ph of 6. CellTiter-Glo A CellTiter-Glo assay checks for cell viability by using luminescence to measure ATP levels. Next, 5,000 cells were plated in opaque walled 96 well plates. Cells were treated with drug for 24, 48, and 72 hours. Mock included DMSO at 2µL/mL, KPT-330 was at 1µM, Nutlin was used at 10µM. At the desired time point Promega Cell Titer-Glo 2.0 reagent was added and the plate was read with a Synergy 2 Multi-mode reader (BioTek) for luminescence levels. Values were normalized to mock at each time point. 15

17 RESULTS P53 induction by KPT-330 and Nutlin Figure 8: Cal51 cells in Mock treatment at 8 hours (left) and Cal51 in KPT-330 at 16 hours (right). Blue indicates DNA stained with DAPI. Red is p53 immunostained with DO-1 antibody. P53 levels were imaged in Cal51 cells over time to ensure that p53 was responding to the drugs. Approximately 500 cells per condition were measured. P53 levels generally increase over time in cells treated with KPT-330 alone, Nutlin alone, and the combination of the two. Levels of p53 became more varied as time increased in each treatment condition. P53 levels were higher in KPT-330 and Nutlin treated cells than those induced by DMSO alone and Etoposide, a known inducer of p53. There was no statistical significance in the value for SINE at 4 hours. All other values had a p value < Outliers were identified using the ROUT method (Q=1%). 16

18 Figure 9: The range and median of measured fluorescent levels of p53 in individual Cal51 cells. The same was done in MCF7 wild type cells. P values are shown as asterisks on the graph. Two asterisks represents a p value ranging from to 0.1, three asterisks represents a p value < Preliminary data using MCF7 breast cancer cells show results that are consistent with CAL51, particular at later time points of 16 and 24 hours. MCF7 shp53 cells showed no p53 staining only a dim, non-specific cytoplasmic background is observed and no treatment conditions showed any cells with nuclear p53 staining. This MCF7 shp53 cell line has been validated and published previously by R. Agami (Brummelkamp, Bernards, & Agami, 2002). Outliers were identified using the ROUT method (Q=1%). 17

19 Figure 10: Range and median of measured fluorescent levels of p53 in individual MCF7 cells. MCF7 shp53 knock down Figure 11: Staining for p53 using DO-1 antibody at 1 S exposure time in MCF7 wild type cells (left) and MCF7 shp53 cells. Both had been treated with KPT-330 and Nutlin in combination for 8 hours. 18

20 Cell Viability Figure 12: Luminescence levels of ATP in a CellTiter-Glo assay read for MCF7 wild type (top) and shp53 (bottom) cells. Each time point is normalized to the mock at that time. 19

21 In wild type MCF7 cells, KPT-330 reduced cell population to 36.5% of the untreated population by 72 hours. Nutlin alone reduced cell population to 25.15%. The two drugs in combination resulted in 26.15% of the population remaining. There was no statistical significance between mock and KPT-330 at 24 hours. The difference between Mock and Nutlin at 24 hours was very significant, P = , and the difference between Mock and the combination at 24 hours was very significant, P= All other values had P values < Staurosporine (STS) was used in each CellTiter-Glo read as a positive control for death. In MCF7 shp53 cells, at 72 hours KPT-330, Nutlin, and the combination of the two resulted in a remaining 48.34%, 81.75%, and 17.26% of the untreated population, respectively. There was no statistical significance for any of the conditions at any of the 24 hour conditions except for STS, which had a P value < The values for Nutlin at 48 and at 72 hours had no statistical significance in difference from the Mock values. At 48 hours, the value for KPT-330 had a P value of All other values had P values < Senescence Staining with Beta-Galactosidase Wild type MCF7 cells began to show staining after 96 hours of treatment, shp53 MCF7 cells began to show staining at 48 hours. Overall, staining levels were greater in shp53 cells. However, the data still needs quantification. No immediate difference was observed between treatment type in either cell type. 20

22 Untreated SINE Nutlin SINE + Nutlin Figure 13: MCF7 wild type (top row) and shp53 (bottom row) cells stained for Beta-Galactosidase after 96 hours of treatment Cell cycle Phenotypes using FUCCI The number of red to green cells was recorded for one position per condition from timelapse video. At each time point the video was paused and each cell was marked with an ROI in FIJI (NIH). Levels of fluorescence of both GFP and RFP were measured and normalized to calculate the number of cells expressing each color. Each drug treatment showed a general shift to red, resulting in a >90% G1 population in each treatment except for SINE in wild type cells. More positions will be quantified to increase accuracy. 21

23 Figure 14: The proportion of cells in each cell-cycle stage tracked in one position from time lapse at various time points. 22

24 Death was also observed through time-lapse imaging. MCF7 shp53 cells underwent little death when treated with Nutlin, observed in CellTiter-Glo as well. Despite lack of death, these cells show a strong G1 arrest phenotype. Wild type cells treated with KPT-330 and Nutlin had large death and G1 arrest fates. Figure 15: MCF7 shp53 cells in the same position at the time of treatment (left) and 72 hours (right) in SINE treatment. Note the death and that the proportion of red cells that shifts from 61% to 98%. Figure 16: MCF7 shp53 cells in the same position at time of treatment (left) and 72 hours (right) in Nutlin. The population of red cells began at 38% and ended at 97%. 23

25 Figure 17: MCF7 wild type cells in the same position at time of treatment (left) and 72 hours (right) in KPT-330 and Nutlin. The population of red cells began at 74% and ended at 97%. DISCUSSION P53 is induced by KPT-330 Our immunostaining confirmed that p53 is strongly induced by KPT-330 in Cal51 and MCF7 cells that still contain wild type p53. In Cal51 p53 levels rose over time in treatment with KPT-330, Nutlin, and the two combined over time. MCF7 levels seemed to fluctuate more; this will be reevaluated with time-lapse microscopy of a p53-venus reporter in MCF7 cells as well as through another immunofluorescence assay. Since p53 is induced, it is capable of having a strong effect on cell fate. The dynamics of p53 are often correlated with a specific cell fate (Purvis et al., 2012). KPT-330 has a strong effect on wild type and shp53 cells Despite p53 levels only rising in wild type cells, death and arrest were noted in shp53 cells as well. In fact, cells lacking p53 seem to die in a shorter time period after division. Cells also show a stronger senescent phenotype through beta-galactosidase staining. This may suggest 24

26 that p53 attempts to rescue the cell under the stress induced by KPT-330. Cells also show a strong G1 arrest response to KPT-330. The lack of p53 results in the cell committing to a fate in a shorter amount of time. In response to the lack of XPO-1 activity, p53 may be able to induce G1 arrest to repair DNA before the cell attempts to enter S phase and begin replication. SINE also blocks XPO-1 dependent export of several cell cycle regulators, which could result in the cell being unable to enter S phase independent of p53. Since we see this strong effect in wild type and shp53 cells, it is likely that G1 arrest is independent of p53. Whether or not this phenotype will be observed without p73 is still unknown. Nutlin does not kill p53 cells but does result in a cell cycle phenotype We did not observe a strong death response to Nutlin in MCF7 shp53 cells in either the CellTiter-Glo assay or time lapse. Nutlin did however, have a similar death response to KPT-330 in wild type cells. From the time lapse, we know that while Nutlin may not be killing shp53 cells, it is causing a strong G1 arrest phenotype. We can conclude that Nutlin-induced death is dependent on p53, but there are other pathways in the cell that are stressed by Nutlin. This could be due to MDM2 binding of p73, which does not result in degradation, but inactivates the transcriptional function of p73 (Bálint, Bates, & Vousden, 1999). When MDM2 is blocked, it is possible that p73 is able to function and induce arrest, but not death. It is also possible that MDM2 is creating other stress on the cell independent of tumor suppressors, or possibly a combination of the two. Nutlin in combination with KPT-330 Interestingly, although Nutlin alone does not strongly kill shp53 cells, its combination with KPT-330 creates a stronger death phenotype than KPT-330 alone. The combination of the 25

27 two drugs in wild type cells may create a larger rise in p53, but has a similar kill rate to either Nutlin or KPT-330 alone. More death was observed in the time-lapse of wild type cells in Nutlin and KPT-330, and needs further quantification. Nutlin could be inducing similar pathways to those XPO-1 does to strengthen the effect of the drugs, or it may be inducing a separate stress on the cell that is able to finally push the cells over the edge towards death. Nutlin could be holding cells in G1 phase (Fig. 14) and sensitizing to killing by KPT-330, which is known to kill cells in G1 (Marcus et al.). KPT-330 is responsible for sequestering p53 in the nucleus so it is able to act, and Nutlin blocks MDM2 from ubiquitinating p53. Based on the current model, p53 must be exported before it is degraded. If this is the case, Nutlin should not be increasing the ability of p53 since its degradation would already be largely hindered by KPT-330. This theory is currently confirmed by our results from the CellTiter-Glo assay, yet there appears to be more death in time-lapse videos. Only about 90% of XPO-1 proteins are blocked by maximum doses of SINE drugs, which means that Nutlin could be responsible for inhibiting the degradation of p53 that would result from 10% functional XPO-1 (Crochiere et al., 2016). Currently the most compelling theory is that Nutlin induces an overall stress response in the cell, further initiating tumor suppressors. This theory will need much more testing to confirm. 26

28 FURTHER DIRECTIONS Our plans to move forward with this project begin with creating a p73 or p21 knock-out MCF7 lines and a p53-p73 dual knock-out line. Using these lines we will be able to observe whether or not p73 is able to compensate for p53 entirely when treated with KPT-330, or if an unrelated pathway is at play in the cell. P21 will also be analyzed for its role in the cellular response to SINE drugs. A matched pair HT1080 p53+ and p53- line was recently created in the lab using the CRISPR/Cas9 system and will tested to insure that the phenotypes we are observing are universal to all cell lines. We also have a HCT116 matched pair in which the p53- line is a genetic null. This line will establish that the difference we are seeing in cell fate is not affected by any remaining p53, which may not have been knocked down completely. We want to understand how the death we are observing is being induced, and intend to test the cells with markers for Mitochondrial Outer Membrane Permeabilization (MOMP), an apoptosis event, and in a cell line with increased levels of BCL2, which protects from apoptosis. We can draw more conclusions about which pathways are being induced and by which proteins if we know how the cells are ultimately dying. In the future we plan to do transcriptional analysis of cells treated with KPT-330, Nutlin, and the combination of the two. P53 exerts an effect on the cell through transcriptional regulation. If we observe the strongest change in regulation to be in targets of p53, it will be obvious that p53 is predominantly choosing the fate of the cell. Transcriptional analysis will give us the opportunity to look for protein upregulation that we have not expected, and point at other players in the response to KPT

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