High-throughput testing in head and neck squamous cell carcinoma identifies agents with preferential activity in human papillomavirus-positive or negative cell lines.

Head and neck squamous cell carcinoma (HNSCC) is a common cancer diagnosis worldwide. Despite advances in treatment, HNSCC has very poor survival outcomes, emphasizing an ongoing need for development of improved therapeutic options. The distinct tumor characteristics of human papillomavirus (HPV)-positive vs. HPV-negative disease necessitate development of treatment strategies tailored to tumor HPV-status. High-throughput robotic screening of 1,433 biologically and pharmacologically relevant compounds at a single dose (4 μM) was carried out against 6 HPV-positive and 20 HPV-negative HNSCC cell lines for preliminary identification of therapeutically relevant compounds. Statistical analysis was further carried out to differentiate compounds with preferential activity against cell lines stratified by the HPV-status. These analyses yielded 57 compounds with higher activity in HPV-negative cell lines, and 34 with higher-activity in HPV-positive ones. Multi-point dose-response curves were generated for six of these compounds (Ryuvidine, MK-1775, SNS-032, Flavopiridol, AZD-7762 and ARP-101), confirming Ryuvidine to have preferential potency against HPV-negative cell lines, and MK-1775 to have preferential potency against HPV-positive cell lines. These data comprise a valuable resource for further investigation of compounds with therapeutic potential in the HNSCC

Gene expression profiles and biomarker identification for KMT5A identifies novel potential therapeutic targets in prostate cancer

PhD ThesisProstate cancer (PC), is initially androgen dependent due to the androgenic nature of the organ. Hence, initial therapy comprises androgen depletion via chemical castration in conjunction with an anti-androgen therapeutic. However, patients relapse and the tumours aggressively re-grow in a castrate resistant (CRPC) manner. In CRPC, androgen receptor (AR) signaling remains functional via numerous mechanisms hence the AR remains a viable therapeutic target. However, treatment with current AR targeting therapeutics also results in relapse indicating the potential of targeting AR signaling indirectly by targeting AR co-factors. Recently, KMT5A, a lysine methyltransferase, has been identified as an AR co-activator exclusively in models of CRPC. A number of KMT5A inhibitors have been identified recent years, which would enhance the possibility of targeting KMT5A in PC. This thesis aims to determine the signature of genes that are regulated directly by KMT5A or by combined activities of AR and KMT5A in PC cell lines and to further identify biomarkers for KMT5A activity. These aims were approached using Illumina Human HT-12 arrays to detect KMT5A gene expression profiles in an in vitro cell line model of androgen independent PC (LNCaP-AI cells). Microarray data analysis revealed a number of androgenregulated genes to be modulated by KMT5A concurrently, and other genes that were found to be regulated by KMT5A activity, and a further cohort of genes that were found to be regulated solely by KMT5A. CDC20 was selected for further study from the identified KMT5A regulated genes as a possible biomarker for KMT5A activity in aggressive PC. KMT5A was found to regulate CDC20 mRNA and protein expression. The enzymatic activity of KMT5A was demonstrated to affect CDC20 expression through the enrichment of the H4K20me1 mark at the CDC20 promoter in androgen-sensitive (LNCaP) and androgen-independent (LNCaP-AI) cells. The regulation of CDC20 by KMT5A expression, therefore identifies CDC20 as putative biomarker for KMT5A activity. KMT5A was also shown to influence CDC20 expression via p53. Knockdown of KMT5A inhibited the mono-methylation of p53 at K382 to enhance p53 activity, demonstrated by increased p21 expression which negatively regulated CDC20 ii expression. These findings were confirmed using commercially available KMT5A inhibitors Ryuvidine and UNC0379. In summary, KMT5A inhibition in PC cells using small molecule inhibitors may provide benefit to patients that have relapsed on AR- targeting therapeutics and as such requires further investigation as a potential therapeutic target. CDC20 was identified as a putative biomarker for KMT5A activity which may prove useful to detect effective KMT5A inhibition in these studies.The Higher Committee for Education Development in Ira

TIMMA-R : an R package for predicting synergistic multi-targeted drug combinations in cancer cell lines or patient-derived samples

Network pharmacology-based prediction of multi-targeted drug combinations is becoming a promising strategy to improve anticancer efficacy and safety. We developed a logic-based network algorithm, called Target Inhibition Interaction using Maximization and Minimization Averaging (TIMMA), which predicts the effects of drug combinations based on their binary drug-target interactions and single-drug sensitivity profiles in a given cancer sample. Here, we report the R implementation of the algorithm (TIMMA-R), which is much faster than the original MATLAB code. The major extensions include modeling of multiclass drug-target profiles and network visualization. We also show that the TIMMA-R predictions are robust to the intrinsic noise in the experimental data, thus making it a promising high-throughput tool to prioritize drug combinations in various cancer types for follow-up experimentation or clinical applications.Peer reviewe

CDC20 Is Regulated by the Histone Methyltransferase, KMT5A, in Castration-Resistant Prostate Cancer

The methyltransferase KMT5A has been proposed as an oncogene in prostate cancer and therefore represents a putative therapeutic target. To confirm this hypothesis, we have performed a microarray study on a prostate cancer cell line model of androgen independence following KMT5A knockdown in the presence of the transcriptionally active androgen receptor (AR) to understand which genes and cellular processes are regulated by KMT5A in the presence of an active AR. We observed that 301 genes were down-regulated whilst 408 were up-regulated when KMT5A expression was reduced. KEGG pathway and gene ontology analysis revealed that apoptosis and DNA damage signalling were up-regulated in response to KMT5A knockdown whilst protein folding and RNA splicing were down-regulated. Under these conditions, the top non-AR regulated gene was found to be CDC20, a key regulator of the spindle assembly checkpoint with an oncogenic role in several cancer types. Further investigation revealed that KMT5A regulates CDC20 in a methyltransferase-dependent manner to modulate histone H4K20 methylation within its promoter region and indirectly via the p53 signalling pathway. A positive correlation between KMT5A and CDC20 expression was also observed in clinical prostate cancer samples, further supporting this association. Therefore, we conclude that KMT5A is a valid therapeutic target for the treatment of prostate cancer and CDC20 could potentially be utilised as a biomarker for effective therapeutic targeting

CDC20 is regulated by the histone methyltransferase, KMT5A, in castration resistant prostate cancer

The methyltransferase, KMT5A has been proposed as an oncogene in prostate cancer and therefore represents a putative therapeutic target. To confirm this hypothesis we have performed a microarray study in a prostate cancer cell line model of androgen independence following KMT5A knockdown in the presence of transcriptionally active androgen receptor (AR) to understand which genes and cellular processes are regulated by KMT5A in the presence of an active AR. We observed that 301 genes were down-regulated whilst 408 were up-regulated when KMT5A expression was reduced. KEGG pathway and Gene Ontology analysis revealed apoptosis and DNA damage signal- ing are up-regulated in response to KMT5A knockdown whilst protein folding and RNA splicing were down-regulated. Under these conditions, the top non-AR regulated gene was found to be CDC20, a key regulator of the spindle assembly checkpoint with an oncogenic role in several cancer types. Further investigation revealed that KMT5A regulates CDC20 in a methyltransferase depend- ent manner to modulate both histone H4K20 methylation within its promoter region and indirectly via the p53 signalling pathway. A positive correlation between KMT5A and CDC20 expression was also observed in clinical prostate cancer samples further supporting this association. Therefore, we conclude that KMT5A is a valid therapeutic target for the treatment of prostate cancer and CDC20 could potentially be utilized as a biomarker for effective therapeutic targeting

THE IDENTIFICATION AND CHARACTERIZATION OF PROTEIN KINASE INHIBITORS TARGETING BREAST CANCER STEM CELLS

Breast cancer is the most common cancer among Canadian women with one in nine women expected to develop breast cancer in their lifetime. Until recently these breast tumors were thought to be a homogeneous cell population. Recent studies have shown that breast tumors contain a rare cell type termed breast tumor initiating cells (TICs) or cancer stem cells (CSCs) with the ability to elicit new tumor growth and metastases. These TICs exist apex of a tumor cell hierarchy and give rise to more TICs and non-tumorigenic cells. Traditionally, drugs were developed to target the highly proliferative cells population resulting in a decrease in tumor volume. However, these therapies spare the TICs, which results in tumor relapse demonstrating the need for new drugs that target the TICs. Because in cancer, mutated protein kinases are the controllers of cell proliferation, invasion and metastasis, they have become a target for drug development. Inhibition of these kinases could lead to the identification of compounds that selectively target breast TICs. Using mammary tumors from cancer prone mice propagated as non-adherent tumorspheres (TMS), which contain a high fraction of breast TICs and the same conditions to propagate the non-transformed mouse mammary epithelial stem and progenitor cells (MESC), as non-adherent mammospheres (MMS) a 240-kinase inhibitor library was screened using an AlamarBlue proliferation assay. Twenty percent of the compounds resulted in 75% decrease in proliferation of TMS derived cells and some of which were TMS-selective. Sunitinib, a multi-targeted kinase inhibitor, was one of the selective compounds identified and when administered to mice with subcutaneous mammary tumors resulted in tumor shrinkage. This was accompanied by an increase in apoptotic cells, decrease in proliferating cells and tumor vasculature, and a change in tumor morphology and composition. These findings show the efficacy of Sunitinib in shrinking mouse mammary tumors and suggest a potential use of Sunitinib for treatment of breast cancer.Master of Science (MSc

Modulación epigenética en el tratamiento personalizado del glioblastoma

Introducción: El glioblastoma es el tumor maligno del sistema nervioso central más común y con peor pronóstico. A pesar del tratamiento con Temozolomida la supervivencia es muy baja y las recidivas ocurren en la mayoría de los casos. La epigenética permite comprender la tumorigénesis y predecir la respuesta al tratamiento oncológico, así como constituir dianas terapéuticas. Objetivos: Los objetivos del estudio son definir el patrón de resistencia/sensibilidad en base a viabilidad celular para cada cultivo procedente de pacientes frente a un panel de fármacos con efectos a nivel epigenético y validar el efecto de los fármacos más prometedores mediante una curva dosis-respuesta. Metodología: Se trata de un estudio experimental realizado en cultivos procedentes de pacientes diagnosticados de glioblastoma en el que se estudia la acción de los fármacos incluidos en el panel Tocriscreen Epigenetics Library (número de catálogo 6801) midiendo la viabilidad celular con el reactivo AlamarBlue HS. Resultados: Se estudia la acción del panel de fármacos obteniendo diferentes perfiles de sensibilidad farmacológica para cada línea celular de glioblastoma y destacando la acción de hidrocloruro de hesperadina y ryuvidina. Posteriormente se valoran estos compuestos mediante curvas dosis-respuesta demostrando que las sensibilidades farmacológicas son dependientes de la concentración. También se valoran los cambios morfológicos apreciando diferencias considerables a medida que aumenta la concentración. Conclusiones: Cada línea celular responde de manera diferente a los moduladores epigenéticos por lo que el tratamiento personalizado puede ser una opción terapéutica esperanzadora. Además, hidrocloruro de hesperadina parece ser una de las opciones más interesantes.Introduction: Glioblastoma is the most common malignant tumour of the central nervous system with the worst prognosis. Despite treatment with Temozolomide, survival is very low and recurrences occur in most cases. Epigenetics allows understanding tumorigenesis and predicting response to oncological treatment, as well as constituting therapeutic targets. Objectives: The objectives of the study are to define the pattern of resistance/sensitivity based on cell viability for each culture from patients against a panel of drugs with effects at the epigenetic level and to validate the effect of the most promising drugs by a dose-response curve. Methodology: This is an experimental study performed on cultures from patients diagnosed with glioblastoma in which the action of the drugs included in the Tocriscreen Epigenetics Library panel (catalog number 6801) is studied by measuring cell viability with the AlamarBlue HS reagent. Results: The action of the panel of drugs was studied obtaining different pharmacological sensitivity profiles for each glioblastoma cell line and highlighting the action of hesperadin hydrochloride and ryuvidine. Subsequently, these compounds are evaluated by means of dose-response curves demonstrating that pharmacological sensitivities are concentration-dependent. Morphological changes are also assessed appreciating considerable differences as concentration increases. Conclusions: Each cell line responds differently to epigenetic modulators so personalized treatment may be a hopeful therapeutic option. Moreover, hesperadin hydrochloride seems to be one of the most interesting options

Development of a Direct Activity Probe for Rho-Associated Protein Kinase

Hepatocellular carcinoma (HCC) is an extremely aggressive form of liver cancer with a low survival rate due to high recurrence. Increases in Rho-associated Protein Kinase (ROCK) activity are correlated with a more aggressive, metastatic phenotype associated with advanced HCC. Inhibitors for ROCK have shown potential for the reduction of this metastatic phenotype of HCC. We outline the design and optimization of a direct activity sensor for ROCK that utilizes a phosphorylation-sensitive sulfonamido-oxine fluorophore, termed Sox, and is capable of reporting on the inhibition of ROCK. This CSox-based activity probe utilizes chelation-enhanced fluorescence (ex. = 360 nm and em. = 485 nm) between the proximal phosphorylated residue, Mg2+ and the Sox fluorophore. This allows for the direct and continuous monitoring of phosphorylation of the peptide-based probe over time. The sensitivity of the optimal CSox-based probe, ROCK-S1, was detected to be 10 pM of recombinant enzyme. Using this probe we demonstrate the ability to directly and rapidly assess a pilot small molecule library for inhibitors of ROCK2, using a robotics platform. In a step towards applying our probe in complex biological systems, we identify the optimal conditions for monitoring ROCK2 while inhibiting off-target enzymes (PKCα, PKA, and PAK). Our work provides a sensitive assay platform for ROCK activity that is compatible to HTS and could potentially be used to interrogate ROCK activity in heterogeneous biological samples. Advisor: Cliff I. Stain