The work described in this thesis profiled the transcriptome of undifferentiated (TSCs) and 4-days differentiated (TGCs) cells through RNA-seq and identified ~7,800 differentially expressed genes which include regulators of the cell cycle, apoptosis, cytoskeleton, cell mobility, embryo implantation, metabolism and various signaling pathways. We revealed that several mitotic proteins, including Aurora A kinase, were downregulated in TGCs and that the activity of Aurora A kinase is required for the maintenance of TSCs. We also identified hitherto undiscovered, cell-type-specific alternative splicing events in 31 genes and expression of 19 novel exons in 12 genes in TSCs and TGCs. Similarly, we discovered that the expression of Cyclin-dependent Kinase 1 (Cdk1) was downregulated in fused BeWo cells and its inhibition through small-molecule inhibitor induced fusion in these cells. The role of Caspase3 downregulation in conferring resistance to genotoxic stress-induced apoptosis in developmentally programmed polyploid cells was also investigated. Finally, we discovered that the polyploid nuclei in fused BeWo cells result from nuclear fusion following the fusion of the cells. Overall, results described in this dissertation uncover several potential regulators of TSC differentiation and TGC function, thereby providing a valuable resource for developmental and molecular biologists interested in the study of stem cell differentiation and embryonic development. Moreover, these results will help in understanding the role and regulation of apoptosis in differentiated TGCs.

Publications: 

Riaz M.R., Preston G., Mithani A.* (2020) MAPPS: A web-based tool for metabolic pathway prediction and network analysis in the post-genomic era. ACS Synthetic Biology. 9(5):1069–1082  

Publication:

Ahsan A, Khan AZ, Javed H, Mirza S, Chaudhary SU, Shahzad-Ul-Hussan S.  Estimation of hepatitis C prevalence in the Punjab province of Pakistan: A retrospective study on general population.  PLoS One. 2019 Apr 3;14(4):e0214435. doi: 10.1371.

Publication:

Umer, Z*., Akhtar, J*., Khan, MHF*., Shaheen, N., Haseeb, MA., Mazhar, K., Mithani, A., Anwar, S and Tariq, M. (2019). Genome-wide RNAi screen in Drosophila reveals Enok as a novel Trithorax group regulator. Epigenetics and Chromatin 12, 55 (2019) doi:10.1186/s13072-019-0301-x

Publication:

Umer, Z*., Akhtar, J*., Khan, MHF*., Shaheen, N., Haseeb, MA., Mazhar, K., Mithani, A., Anwar, S and Tariq, M. (2019). Genome-wide RNAi screen in Drosophila reveals Enok as a novel Trithorax group regulator. Epigenetics and Chromatin 12, 55 (2019) doi:10.1186/s13072-019-0301-x

 *: Equal authors

Abstract:

1. Identification and evaluation of novel therapeutic strategies targeting oral cancer in Pakistan 

Oral cancer is the most prevalent subtype of head and neck cancers that arises mainly from squamous cells of the oral cavity. It is the most common type of cancer in males and the second most common among females in Pakistan and accounts for 10% of all malignancies compared to 4% worldwide. The work described in this thesis identifies novel therapeutic targets against oral cancer. First, the growth of oral cancer cell lines of Asian origin and primary tumor cells isolated from oral cancer patients was optimized in 2D monolayer and 3D spheroid/organoid cultures for evaluating various targeted drugs and their combinations. Twelve small molecule inhibitors targeting key cellular proteins were initially evaluated in oral cancer cell lines for their antiproliferative activity. This resulted in the identification of pan aurora kinase inhibitor CCT137690, which caused abrogated mitotic spindle formation, misaligned chromosome attachment and polyploidy, leading ultimately to apoptotic cell death. CCT137690 also synergized with gefitinib (EGFR inhibitor) and pictilisib (PI-3 kinase inhibitor) in oral cancer cells. Drugloaded polyethylene glycol (PEG)-based nanocapsules were then synthesized for co-administration of the identified combinations. Nanocapsules loaded with combinations of CCT137690 with gefitinib or pictilisib inhibited the growth of oral cancer cell lines in 3D spheroid cultures and induced apoptosis comparable to the free drug combinations. Furthermore, high throughput screening with a library of 1127 small molecule inhibitors in oral cancer cells identified several potent inhibitors and their synergistic combinations. A combination of carfilzomib (proteasome inhibitor) and YM-155 (survivin inhibitor) exhibited potent antiproliferative activity against all oral cancer cells, including 3D organoid cultures from patient-derived cells. Finally, the mutational profile of patient-derived cells was determined using whole exome sequencing and RNA seq analysis. Together, this data identifies new vulnerabilities of oral cancers to various targeted drug combinations that can be used to devise new treatment strategies for oral cancer patients in Pakistan.

2. Identification and characterization of Aurora Kinase inhibitors.

Aurora kinases (Aurora A, B, and C) are a family of serine/threonine kinases that are ubiquitously expressed, and their overexpression and/or amplification in many cancers have been associated with poor prognosis. Here, we utilized chemical and in silico approaches to identify novel aurora kinase inhibitors. For the first approach, a small library of quinones was screened in a biochemical assay for aurora A kinase resulting in the identification of two compounds, natural quinone naphthazarin (1) and a pseudo anthraquinone, 2- (chloromethyl)quinizarin (11), that potently inhibited the proliferation of various cancer cell lines. Treatment of cancer cells with these compounds for 24 h resulted in abrogated mitosis and apoptotic cell death. Direct binding of both the compounds with aurora A kinase was also confirmed through STD NMR analysis. Docking studies predicted the binding of both compounds to the ATP binding pocket of aurora A kinase. For the second approach, an in silico screening for aurora a kinase inhibitors was performed with a library of 1.8 million compounds; six hits were selected based on their ability to bind with the pharmacophore template. Of the six identified hits, only one inhibitor, 688, was validated to bind to both aurora A and B kinases and inhibit them in cells. Treatment of cells induced polyploidy and abrogated mitosis, ultimately leading to apoptotic cell death. Finally, NMR studies showed that the confirmation of aurora A-bound 688 was similar to the one predicted through docking studies. Hence these studies report the identification and characterization of a novel aurora kinase inhibitor.

The following two additional projects were also part of the thesis.

1. Identification of MYCN targeting compounds for the treatment of MYCN-amplified Neuroblastoma

2. Identification of compounds selectively targeting mutant KRAS-dependent cancers

Publications:

• Furqan M, Fayyaz A, Firdous , Raza H, Bilal A, Saleem RSZ, Hussan SS,Wang D, Youssef FS, Al Musayeib NM, Ashour ML, Hussain H and Faisal A. (2022) Identification and Characterization of Natural and Semisynthetic Quinones as Aurora Kinase Inhibitors. Journal of Natural Products 2022, 85, 6, 1503–1513

• Farrukh UB, Bilal A, Zahid H, Iqbal M, Manzoor S, Firdous F, Furqan M, Azeem M, Emwas AH, Alazmi M, Gao X, Saleem RSZ, Faisal A (2022) Synthesis and Evaluation of Novel Carboxamides Capable of Causing Centrosome Declustering and Apoptosis in Breast Cancer Cells. ChemistrySelect 2022, 7, e202104218 (1 of 7) 

• Tariq MU, Furqan M, Parveen H, Ullah R, Muddassar M, Saleem RSZ, Bavetsias V, Linardopoulos S and Faisal A (2021) CCT245718, a dual FLT3/Aurora A inhibitor overcomes D835Y-mediated resistance to FLT3 inhibitors in acute myeloid leukaemia cells. British Journal of Cancer British Journal of Cancer 2021 125 (7), 966-974

•  Zahra R, Furqan M, Ullah R, Mithani A, Saleem RSZ, Faisal A (2020) A cell-based highthroughput screen identifies inhibitors that overcome P-glycoprotein (Pgp)-mediated multidrug resistance. Plos One 15 (6), e0233993 

• Furqan M, Huma Z, Ashfaq Z, Nasir A, Ullah R, Bilal A, Iqbal M, Khalid MH, Hussain I, Faisal A. (2019) Identification and evaluation of novel drug combinations of Aurora kinase inhibitor CCT137690 for enhanced efficacy in oral cancer cells. Cell Cycle 2019 Sep;18(18):2281-2292 

• Shah OS, Chaudhary FA, Awan HA, Fatima F, Arshad A, Bibi A, Ahmed M, Hameed H, Furqan M, Khalid S, Faisal A, Ullah Chaudhary SU. (2018) ATLANTIS - Attractor Landscape Analysis Toolbox for Cell Fate Discovery and Reprogramming. Scientific Reports 2018 Feb 23;8(1):3554.