Dr. Spiottos’s long-term research goal is to improve the treatment of patients with virally induced
cancers. The Human Papillomavirus (HPV) oncogene E6 degrades and inhibits the tumor
suppressor p53 allowing cells to escape apoptosis (programmed cell death). He hypothesizes that
these E6 inhibitors will be specifically cytotoxic to HPV cancer cells while leaving the normal cells
of patients unharmed. Dr. Spiotto has developed a novel cell based reporter assay to detect the
interaction between E6 and p53.

The preliminary reporter system that Dr. Spiotto initially planned used E6 alone to degrade a
p53 reporter protein. However, when this system was constructed it did not provide a significant
separation in signal between the experimental and control cell populations that would be appropriate
for high throughput screening. Several revisions were made to arrive at a final novel protein
knockdown system that specifically monitors E6 mediated degradation of p53. Additionally, he
has implemented several means within this assay to increase the chance of finding a specific E6
inhibitor, such as optimizing the reporter screen to detect specific E6 inhibitors and selecting for cell
permeable compounds that are less likely to be toxic.

Using the reporter system described above, Dr. Spiotto conducted several screens for compounds
that inhibit the E6-p53 interaction. 77,570 compounds were screened, and 54 compounds were
selected for further testing. Dr. Spiotto will use the E6 specific inhibitors identified in the screen to
determine whether they can restore p53 levels in and inhibit growth of HPV-induced cancer cells
which could eventually translate into clinically useful adjuvant therapies. He is currently gathering
information on the candidate compounds to determine their specific toxicity levels and whether they
increase p53 expression levels in HPV cancer cells. Results of these efforts are expected in the next
few months.

My long-term research goal is to improve the treatment of patients with virally induced cancers. My objective with the Cancer Research Foundation Grant is to develop a new reporter assay for the Human Papillomavirus (HPV) oncogene E6 and use it to identify new compounds that inhibits E6 and restores the p53 pathway in cancer cells. I hypothesize that these E6 inhibitors will be specifically cytotoxic to HPV cancer cells while leaving the normal cells of patients unharmed. My rationale for this work is that it would provide novel lead compounds that I expect to translate into a clinically useful and non-genotoxic drugs that specifically target HPV cancers. To date, the summary of the results for the following Specific Aims is described below: Aim 1: Development of a cell based high throughput (HT) reporter assay for the HPV oncogene E6. The HPV oncogene E6 degrades and inhibits the tumor suppressor p53 enabling cells to escape apoptosis. Here, I will develop a novel cell based reporter assay to detect the interaction between E6 and p53. Progress: The initial reporter system was initially planned to use E6 alone to degrade a p53 reporter protein. While this system was constructed, it did not provide a significant separation in signal between the experimental and control cell populations that would be appropriate for high throughput screening. Therefore, after several revisions, I will detail the final novel reporter assay used for high throughput screening. I have developed a novel cell based reporter assay to interrogate the E6-p53 interaction using a protein knockdown strategy (1, 2). Based on previous strategies, I used E6 as a prey protein to target the bait protein, p53, to the SCF ubiquitin ligase complex for protein degradation (Fig 1). The SCF complex is an E3 ubiquitin ligase that relies on the protein - TRCP to target proteins to this complex. -TRCP consists of two separate domains, the F-Box domain that binds to the SCF complex and the WD40 domain that binds to its substrate proteins. Using this model, I targeted p53 to the SCF complex by fusing a flagged-tagged F-box domain of - TRCP to the E6 protein to generate a TRCP-E6 (TE6) protein. When TE6 binds to p53, it shuttles p53 to the SCF ubiquitin ligase complex which then ubiquitinates Figure 1: A protein knockdown strategy to monitor the E6:p53 interaction. (A) The SCF complex is an E3 ubiquitin ligase which the adapter protein -TRCP binds to with it’s F-box domain. -TRCP recruits target proteins to the SCF complex using its WD40 domain. (B) When E6 replaces the W40 domain of -TRCP, the TE6 fusion proteins recruits p53 for degradation. (C) When E67 replaces the W40 domain of -TRCP, the TE7 fusion proteins recruits Rb for degradation. p53 and targets it for proteosomal degradation. Furthermore, by forcing p53 degradation through the SCF complex, this strategy avoids identifying inhibitors that target the E6AP ubiquitination pathway. The TE6 gene cassette preceded an internal ribosomal entry site (IRES) driven dsRED fluorescent protein to non-invasively monitor cells expressing TE6 expression. To monitor p53 levels, I fused p53 to firefly luciferase creating the p53-Luc gene. As a control for p53-Luc degradation, the p53-Luc gene preceded an IRES-Enhanced Green Fluorescent Protein (EGFP) gene that served as a marker for p53-Luc transcription and translation in the presence of specific p53-Luc protein degradation. For a specificity control, I used a similar approach to target the E7-Rb interaction. I fused E7 to -TRCP (TE7) as well as fusing Rb to renilla luciferase (Rb-Ren). The separate renilla luciferase reporter enabled me to monitor both RB-Ren and p53-Luc levels in the same sample. I then generated adenoviruses capable of expressing TE6 (Ad-TE6), p53-Luc (Ad-p53) or a control vector expressing dsRED fluorescent protein (Ad-Red; Fig 2). The adenovirus strategy enabled me to optimize the reporter assay by titrating different ratios of TE6 and p53- Luc proteins. I co-infected HPV-negative cervical cancer cells, C33a, with Ad-TE6 and Ad-p53. Using fluorescence cytometry, I observed that cells infected with both the Ad-p53 and Ad-TE6 had similar fluorescence as control cells infected with both Ad-p53 and Ad-Red (Fig 3). Furthermore, the cells with higher levels of dsRED fluorescence had higher levels of EGFP fluorescence indicating that if TE6 caused loss of p53-Luc activity it not be due to loss of transcription or translation of the EGFP gene, and by proxy, the p53-Luc gene. I then determined the extent to which TE6 targeted p53-Luc for degradation. C33a cells infected with Ad-TE6 and Ad-p53 had decreased luciferase activity compared to cells infected with Ad-TE7 or Ad-Red (Fig. 4). Similarly, C33a cells infected with Ad-TE7 and Ad-Rb-Ren had decreased luciferase activity compared to control cells. To confirm that TE6 caused proteosomal degradation of p53-Luc, I treated cells with the proteosomal inhibitor Velcade. Cells infected with Ad-TE6 and Ad-p53 and treated with Velcade had increased p53-Luc activity. By contrast, RITA, an inhibitor of the p53-MDM interaction, failed to increase p53-Luc activity. Finally, I confirmed that loss of p53-Luc signal by TE6 was due to loss of protein expression using Western analysis. Compared to TE7 or dsRed, p53-Luc cells infected with TE6 had decreased expression of the endogenous p53 as well as the p53-Luc fusion protein. Therefore, I Figure 2: Adenoviral vectors for TE6:p53- Luc reporter assay. (Top) Ad-p53 containing the p53-luciferase reporter fusion protein followed by an IRES-EGFP. (Middle) Ad-TE6 containing a flag-tagged F-box domain of bTRCP fusedto E6 followed by an IRES dsRED reporter. (Bottom) The control Ad-Red vector. Figure 3: TE6 does not affect transcription or translation of p53-Luc reporter. Cells were infected with Ad-TE6, Ad-p53 or Ad-Red and 48h later EGFP and RFP fluorescence was monitored by fluorescence cytometry. The TE6 gene did not affect the transcription or translation of the p53-Luc reporter as measured by EGFP fluorescence. have developed a novel protein knockdown system that specifically monitors E6 mediated degradation of p53. This reporter screen is optimized to detect specific E6 inhibitors for several reasons. First, potential E6 inhibitors could increase luciferase decreases the chances of detecting false positives due to cytotoxicity, luciferase inhibitors or aggregation of the compounds. In addition, compared to fluorescent markers, luciferase reporter bypasses the false positive signals due to fluorescent compounds. Furthermore, compared to in vitro assays, this cell based assay selects for cell permeable compounds that are less likely to be toxic. In addition, this cell-based assay may still detect compounds that target other proteins necessary for the E6:p53 interaction and, therefore, identify additional HPV inhibitors. Finally, in the same well as cells expressing TE6 and p53-Luc, I used cells expressing TE7 and Rb-Ren as a specificity control to minimize the chances of detecting proteasome inhibitors. Thus, I have implemented several means to increase the chances of finding a specific E6 inhibitor. Aim 2: High throughput screening of chemical libraries for compounds that inhibit the E6-p53 interaction. I will use the E6 reporter assay developed in Aim 1 to screen two separate chemical libraries for compounds that inhibit E6 activity. I will then validate potential hits with secondary screens to confirm their activity and specificity for the E6-p53 interaction. Progress: Using the reporter system described above, I conducted several screens for compounds that inhibit the E6-p53 interaction. I screened 77,570 compounds including 640 compounds from a Lopac library, 1364 compounds from the NCI Diversity library, 1120 compounds from the Prestwick library, 2000 compounds from the Spectrum library, 446 compounds from the NIH Oncology Drug Library, 800 compounds from the NCI Open Plate Set and 64,000 compounds from the Chembridge Microformat Library. In a single well, I plated C33a cells infected with Ad-TE6 and Ad-p53 (TE6-p53) as well as cells infected with Ad-TE7 and Ad-Rb (TE7-Rb). During this screening, the average Z’ was 0.43 ± 0.15, which is acceptable for cell-based screens. Figure 4: TE6 promotes proteosomal degradation of the p53-Luc reporter. (A) Cells infected with Ad-p53 and AdTE6 had lower levels of firefly luciferase activity. (B) Cells infected with Ad-Rb and Ad-TE7 had lower levels of renilla luciferase activity. (C) Cells in (A) were treated with the proteasome velcade or the MDM2 inhbitior RITA and luciferase activity was assayed 8h later. Figure 5: TE6 expression caused loss of p53-Luc and endogenous p53 protein levels. Cells were infected with Ad-p53 and Ad-TE6, Ad-Red or Ad-TE7. Lysates were collected 24h later and assayed for p53 expression, luciferase expression or TE6 expression using an anti-FLAG epitope. Figure 6 plots 3841 sample compounds with p53-Luc and Rb-Ren values normalized to baseline. The red region depicts compounds that specifically inhibited the TE6:p53-Luc interaction. I selected compounds based with the following characteristics: compounds were required to increase the p53 signal by at least 3-fold, be at least 5 standard deviations above the untreated control, cause greater than 30% inhibition of the E6:p53 interaction compared to control wells and specifically inhibit the E6:p53 interaction compared to the E7:Rb interaction. Based on these criteria, I selected 54 compounds for further testing. Therefore, I used a novel cell based reporter assay to conduct a high-throughput screen for compounds that specifically inhibited the E6-p53 interaction. I have identified several unique compounds for further testing which I will describe below. Using the following approach, I expect to find non-genotoxic compounds that inhibit HPV oncogenes and would eventually translate into clinically useful adjuvant therapies. Aim 3: Assay E6 inhibitors for effects on endogenous p53 levels and cell survival in HPVinduced cancer cells. I will use E6 specific inhibitors identified in Aim 2 to determine whether they can restore p53 levels in and inhibit growth of HPV-induced cancer cells. Progress: I have validated 29 candidate compounds and have tested them for both selective cytotoxicity and the ability to restore endogenous p53 levels. Figure 7 demonstrates that several of these compounds may restore endogenous p53 levels. Figure 8 demonstrates that of these compounds several also were cytotoxic for the HPV cell line HeLa but not for non-HPV murine embryonic fibroblasts. Figure 6: High throughput screening using the TE6:p53-Luc reporter detects specific E6 inhibitors. A representative plot of 3841 compounds used to treat cells infected with Ad-TE6 and Ad-p53 or Ad-TE7 and Ad-Rb. Compounds were normalized for p53-Luc and Rb-Ren activity. The red area indicates potential E6 ispecific inhibitors. Figure 7: Several hits restored p53-Luc activity and protein expression. (Right panel) 5 validated hits selectively restored p53-Luc activity in C33a cells expressing T-E6. (Left panel) Western analysis of cells treated with validated hits confirm increased p53-Luc activity correlated with increased protein levels. Summary: To date, I have completed the three specific aims. I have obtained funding from the Fanconi Anemia Research Fund as well as the Burroughs Wellcome Fund Career Award for Medical Scientists.