Bowel Disease Research Foundation

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1 Research Project Annual Report Lead investigator Dr Ricky Sharma Institution Department of Oncology, University of Oxford Project Title Making radiotherapy for rectal cancer more effective by identifying which drugs should be used for individual patients Start date Finish date Lay Summary (max 500 words) When they are first diagnosed, over 5000 patients in the UK have rectal cancer that cannot be surgically removed unless it can be shrunk down with radiotherapy. Good versus poor tumour regression grade measured on MRI scan after radiotherapy has been correlated with disease-free survival and overall survival for British patients with locally advanced rectal cancer (Patel et al. 2011). Current standard therapy (which is 5 weeks of radiotherapy plus the chemotherapy drug, 5-fluorouracil, or an oral version of this drug, capecitabine) results in good tumour regression in only 40-50% of patients. This means that half or more of all patients treated with standard chemotherapy and radiotherapy in the NHS are not getting an optimal response to treatment, and they certainly will be getting short- and long-term side effects from treatment such as disturbance of bowel and bladder function, impotence and infertility. The purpose of this project is to find new ways of making radiotherapy more effective to improve survival rates, organ preservation and quality of life. We now routinely test patients with colorectal cancer in NHS clinics for gene defects in their cancer tissue, such as KRAS, BRAF, NRAS and MLH1. In this project, we are screening cancer cells in the laboratory to discover if certain radiosensitising drugs are more effective when gene defects are present in the cancer. In the first 12 months of the project, we have manually developed a highthroughput assay to screen thousands of drugs for their RS activity within a 4-6 week period. The assay has now been automated and shown to have high reproducibility. It can test 4 different drug concentrations per drug per run at a range of different cell numbers to identify compounds with very high and very low radiosensitising potential. Having validated the automated assay, we have already completed screening of 1900 drugs in colorectal cancer cells with a mutation in the BRAF gene. We chose to study this particular gene defect first since patients with cancers characterised by a BRAF mutation have the shortest survival figures of all patients with colorectal cancer. The automated drugs screening for this particular gene defect has now been completed and we are now analysing the results in detail and validating specific hits in order to decide which ones should proceed further into early phase clinical trials.

2 Over the next 12 months, we will use the novel high-throughput assay to study other genes in the same way: PI3K, KRAS and P53. In the clinical trial that results from this project, it is likely that we will need to consider more than one gene for each patient when we decide which drug should be used optimally to enhance radiotherapy in that individual. Background (purpose for project) Patients with rectal cancer currently receive fluoropyrimidine chemotherapy as a radiosensitiser, but this chemo-radiation treatment strategy results in good tumour regression in less than 50% of patients. The applicants find this clinical response rate unacceptable because: 1. Radiotherapy is a very important treatment for rectal cancer. More effective radiotherapy could improve survival rates, organ preservation and quality of life. 2. We now routinely test patients in NHS clinics for gene defects in their cancer tissue, such as KRAS, BRAF, NRAS and MLH1, but we ignore that information for radiotherapy planning. 3. There is scientific evidence from cancer cells grown in the laboratory that certain radiosensitising drugs are more effective when gene defects are present in the cancer (i.e. certain gene defects could represent an Achilles Heel for certain cancers treated with radiotherapy if the right drug is used in combination). 4. We have shown that non-cancer drugs, such as the anti-infection drug, nelfinavir, can make radiotherapy significantly more effective (Qayum et al. 2009). We completed a clinical trial of nelfinavir plus radiotherapy in 10 patients with rectal cancer to demonstrate the safety of this combination (Hill EJ et al, submitted for presentation at NCRI meeting, Liverpool, November 2014). The aim of this project is to develop a high-throughput screening (HTS) technique to identify which drugs should be used for radiosensitisation in the context of single gene mutations in colorectal cancer (CRC). Such an assay does not currently exist and it would allow personalisation of drugs used with radiotherapy tailored to each individual patient. Introduction At the start of the current project, we performed manual screens of the 97- compound US NCI Approved Oncology Library in colorectal cancer cells. Screens were performed in triplicate and data were consistent within and between screens. Positive controls within the screen were reproducibly identified. Data analysis for each drug concentration was normalised using Z scores (Figure 1). In this project, we have proceeded to develop a robotic high-throughput

3 robotic assay which can be used to screen thousands of drugs for their RS activity. The assay has very high reproducibility, and it is not limited by the plating efficiency of cell lines. Importantly, the assay can test 4 different drug concentrations per drug per run, at a range of different cell numbers to identify compounds with very high and very low radiosensitising potential. Methods We developed a radiosensitisation assay for application in an automated high throughput screen (HTS) in colorectal cancer (CRC) cell lines with single gene mutations. Cancer cells with single BRAF mutation V600E were irradiated 6 hours after being treated with drug and allowed to grow for five doubling times (Figure 2). Cell viability was compared between the irradiated and nonirradiated control plates. In total, four colorectal cancer cell lines isogenic for V600E mutation in BRAF (2 pairs wild-type, or with the activating mutation V600E) were screened twice with 1600 FDA approved drugs and 300 FDA approved cancer drugs. Due to the large database, small number of replicates and novelty of the HTS protocol, different statistical approaches were used to select the best method for analysing the data. Raw data were normalized using both plate mean and negative controls, and quality plots contrasted. Selection of candidate hits based on rank product analysis was found to be the best statistical method fitting for our purpose, as this has been shown to be most powerful with a small number of replicates. Probability of false discovery was computed by permutations, with N=100. Several heatmaps were generated for positive controls and drugs based on differences in ΔZ score between irradiated and non-irradiated plates among cell lines or/and drug concentrations to facilitate identification of the top hits (Figure 3). Heatmaps were generated based on false discovery of 5%, 10% and 30% due to the small number of replicates. Results and discussion The PI3K inhibitor PI103 and the HDAC inhibitor vorinostat (SAHA) were shown to be the optimal positive controls for the HTS. Replicate screens correlated generally very well with a mean correlation of Average Z factors were 0.58 and 0.53 for irradiated and non-irradiated plates respectively. Normalization to the mean was more stable and thus chosen for subsequent analyses. Positive controls were consistently top hits, which validated the screen (Figure 3). Delta-Z scores were 2 for positive controls (P 0.05) which were very comparable with previous results from the manual assays. Results for vorinostat (SAHA) were validated by statistically significant results from clonogenic survival experiments performed manually (Figure 4). This demonstrates how hits from the HTS will be validated in vitro, prior to proceeding to translation to other preclinical models and early phase clinical trials. PI3K and MAPK pathways are the two main cellular survival pathways. Activation of these two pathways as well as dysfunctional tumour suppressor

4 protein P53 have been found in many types of cancer including colorectal cancer and has been associated with poor response to DNA damaging agents and ionising radiation (McCubrey, Steelman et al. 2012). Radiotherapy may be improved by modulating DNA repair or signal transduction pathways such as the MAPK or PI3K pathways (Begg et al, Elshaikh et al). Mutations of the key components the MAPK and PI3K pathway, KRAS/BRAF and PIK3CA as well as TP53 mutations are among the most common mutations in colorectal cancer and they are found in 55%, 15% and 59% of colorectal tumours respectively. We now routinely test patients in NHS clinics for gene defects in their cancer tissue including KRAS, BRAF and NRAS. In addition, the mutational status of other genes such as PIK3CA and TP53 can be assessed in tumour biopsies taken from patients at the time of the diagnosis. In parallel with the HTS, we are therefore optimising and validating the assessment of the same genes in the Pathology Department of the Oxford University Hospitals NHS Trust. This will allow rapid translation of results into a clinical trial in patients with specific mutations in their cancer. Conclusions The purpose of this project is to find new ways of making radiotherapy more effective to improve survival rates, organ preservation and quality of life. In this project, we have established a new laboratory method to discover if certain radiosensitising drugs are more effective when gene defects are present in the cancer. In the first 12 months of the project, we have developed an automated, highthroughput assay to screen thousands of drugs for their radiosensitising activity. Having validated this assay, we have completed screening of 1900 drugs in colorectal cancer cells with the V600E mutation in the BRAF gene. We chose to study this gene defect first since patients with cancers characterised by this mutation have the worst prognosis of all patients with colorectal cancer. We have completed drug screening of and we are now analysing the results to discover which are the most suitable radiosensitisers for patients whose cancer contains this particular gene defect. We will now proceed to use the assay to study 3 other gene mutations commonly detected in colorectal cancer. Recommendations for future work Over the next 12 months, we will study the following genes: PI3K, KRAS and P53 with the same 1900 drug library. We have started manual screens of four HCT116 colorectal cancer cell lines isogenic for common mutations in colorectal cancer (PI3K (H1047R), KRAS (G13D) and P53) in order to optimise the planned HTS with 1600 approved non-cancer drugs and 300 cancer drugs. While we are performing this screen, we validate the most promising results from the BRAF gene mutation screen by manual clonogenic survival assays and in other preclinical models.

5 In collaboration with the biostatisticians, we are also developing a novel statistical method, which may offer an effective and practical tool for analysing high-throughput screening data to accelerate the translation of results from this type of project into early-phase clinical trials. In parallel to the laboratory work, we are optimising and validating the assessment of the same genes in the Pathology Department on human tissue samples. This will allow rapid translation of results into a clinical trial in patients with specific mutations in their cancer. In the clinical trial that results from this project, it is likely that we will need to consider more than one gene for each patient when we decide which drug should be used optimally to enhance radiotherapy in that individual. Ulitmately, this project may change clinical practice from a one size fits all radiotherapy treatment approach to the prescription of a drug with radiotherapy based on the genetic profile of the cancer for a more effective treatment. References (author, title, date of publication) Bentzen, S. M. (2008). "From cellular to high-throughput predictive assays in radiation oncology: challenges and opportunities." Semin Radiat Oncol 18(2): Inglese, J., C. E. Shamu, et al. (2007). "Reporting data from high-throughput screening of small-molecule libraries."nat Chem Biol 3(8): Katz, D., E. Ito, et al. (2009). "On the path to seeking novel radiosensitizers." Int J Radiat Oncol Biol Phys 73(4): Kleiman, L. B., A. M. Krebs, et al. (2013). "Comparative analysis of radiosensitizers for K-RAS mutant rectal cancers." PLoS One 8(12): e McCubrey, J. A., L. S. Steelman, et al. (2012). "Ras/Raf/MEK/ERK and PI3K/PTEN/Akt/mTOR cascade inhibitors: how mutations can result in therapy resistance and how to overcome resistance." Oncotarget 3(10): Patel, U. B., F. Taylor, et al. (2011). "Magnetic resonance imaging-detected tumor response for locally advanced rectal cancer predicts survival outcomes: MERCURY experience." J Clin Oncol 29(28): Qayum, N., R. J. Muschel, et al. (2009). "Tumor vascular changes mediated by inhibition of oncogenic signaling." Cancer Res 69(15): Hughes, J. P., S. Rees, et al. (2011). "Principles of early drug discovery." Br J Pharmacol 162(6):