Literature Review Cancer is a major public health problem and continues to be one of the leading causes of death (Heron). Cancer-related illnesses are complex and are a result of the malfunctioning of genes that are responsible for regulating cell growth division and death (Sudhakar). This gives rise to uncontrolled growth and proliferation of cancer cells in the body. Cancer is a multidisciplinary focus medical research as scientists continue to monitor cancer and further develop effective prevention strategies (Hermann). One of the main focuses in cancer research is acquired resistance, as it is a major impediment in tackling cancer. Acquired resistance is a result of mutations in cancer cells that develop resistance, after initial therapy to anti-cancer treatments (Zahreddine). Although acquired resistance is a major hurdle in treating and defeating cancer, there are other factors that also serve as obstacles in overcoming cancer such as intrinsic resistance, multi-drug resistance, and the various molecular mechanisms that can cause resistance. In order to overcome the inexorable rise of resistance, researchers have conducted studies to further investigate the potential use of combination therapies and personalized medicine. Cancer is a result of growth of abnormal cells in the body, and the abhorrent number of cells are capable of invading surrounding tissues (Pubmed link). Behind cardiovascular diseases, cancer is the second leading cause of death, and continues to affect people worldwide (Siegl). Cancer cells continuously grow and proliferate uncontrollably by mitosis, and these dysregulated cells eventually accumulate and develop into heterogenetic tumours (Sudhakar). If cancer cells break away from the primary tumour, they can spread throughout the body and this can result in metastasis (Cooper). The development of cancer can arise from any type of cell in the body giving rise to a diverse number of cancers, and their responses towards treatments can differ. Tumours may also be composed of a heterogenous population of cells amassing genetic and epigenetic changes that contribute to resistance (Zahreddine). Chemotherapy and traditional cytotoxic drugs constitute the backbone in most treatment regiments against cancer. However, their success and effectiveness against cancer are limited due to the inexorable rise of drug resistance (Holohan). Scientists had to explore the diverse molecular mechanisms that underlie cancer growth, and this led to the development of molecularly targeted therapies due to the disease specific-mechanisms that are absent in normal cells (Zahreddine).Many of these treatments are used in the clinic, and patients demonstrated responses to targeted anti-cancer therapies. Nevertheless, cancer cells cease to react to treatment, and the development of drug resistance continues to be a major problem (Zahreddine). There are two types of drug resistance that can emerge: acquired resistance and intrinsic/primary resistance. Both acquired and intrinsic resistance can arise due to various mechanisms involving alterations to drug metabolism or drug target modifications (Zahreddine). Studying drug metabolism, the most studied mode of resistance, involves analysing the uptake, efflux, and detoxification (Zahreddine). Anti-cancer drugs will enter a cell based on its chemical nature, usually they target receptors and transporters to transmit their effects or gain cellular entry, respectively (Gottesman). Reductions of available transporters or mutations that modify transporters (Zahreddine). Other modes of resistance can include: drug inactivation, oncogene addiction, amplification of alternative oncogenes, and or inactivation of alternative survival pathways (Zahreddine). Once resistance becomes present in cancer, treating it becomes problematic (Freidman). Resistance is not limited issue against one drug, it can occur in a multitude of treatments and give rise to multi-drug resistance (Ullah). To overcome cross resistance, combined synthetic lethality studies have studied interactions between EGFR (epidermal growth factor receptor) and PARP inhibition in human triple negative breast cancer cells (TNBC) using a combination of lapatinib and PARP inhibitor ABT-888 (Nowsheen). Triple negative breast cancer is aggressive type of breast cancer, and it has been found to be resistant to standard anti-cancer therapeutics such as angiogenesis inhibitors, EGFR targeted treatments, src kinase, and mTOR inhibitors (Nowsheen, Chacon). Lapatinib would induce a transient DNA repair deficit, increasing cytotoxicity to PARP inhibitor ABT-888. The drug reduced the presence of nuclear BRCA1 and EGFR, induced subcellular localization, and diminished HR-mediated DNA repair (Nowsheen). The results from the study have shown that Lapatinib combined with ABT-888 induced the intrinsic pathway of apoptosis and weakened the survival fraction of TNBC cells. Patients can have an initial response to anti-cancer treatments, but with acquired resistance, they can stop responding to prolonged treatments and thus be responsible for cancer relapse (Housman). Mutations in a population of cells can arise in acquired resistance because they carry a selection benefit, the treatment given to a patient will eradicate the sensitive clones, but select the resistant ones. (Freidman). It has been proposed that a more aggressive treatment would reduce the likelihood of mutations emerging (Housman). However, initial treatment will reduce the overall tumour size, but the remaining population of cells can mutate and even acquire de novo resistance as a result of the anti-cancer treatment (Frediman). However, patients are always at risk for developing resistance throughout any point of treatment (Zahreddine). Experimental evidence has demonstrated how anti-cancer treatments can give rise to acquired resistance. MDM2 inhibitors, such as nutlin-3, have been shown to give rise to p53-mutated multi-drug resistant cancer cells, and it is also a non-genotoxic p53 activator (Michaelis). p53 is a tumour suppressor protein, and is responsible for DNA repair, and inhibition of the cell cycle, it can also induce apoptosis in response to genotoxic stress (Michealis). TP53 gene encodes for p53, and has been found to be the most frequently mutated gene in cancer (Michaelis). It was previously thought that Nutlin-3 would decrease genomic instability, reduce formation of resistance cells, and avoid targeting the DNA integrity. It has also been known that Nutlin-3 to activate p53 responses, as it blocks the interaction between p53 and its inhibitor MDM2 (Brown). In this study, nutlin-3 induced resistance formation was observed in a panel of neuroblastoma, rhabdomyosarcoma, and melanoma cells. The cell panel was chosen for this study as these cancer cells have been known to have low frequencies of p53 mutations, and nutlin-3 had also displayed anti-cancer effects against these cancer cells (Michaelis). Only 2 out of 28 melanoma, neuroblastoma, and rhabdomyosarcoma cells lines adapted to a range of cytotoxic anti-cancer drugs displayed p53 mutations (Michaelis). This demonstrates that the induction of p53 mutations is an exclusive property of nutlin-3. However, in experiments using single p53-wild type cell derived UKF-NB-3 clones it was shown that nutlin-3 induces de novo p53 mutations, and the drug does not select pre-existent p53-mutations in sub-populations that were already present in the original cell lines (Michaelis). The results in the study have demonstrated that nutlin-3 treatment reproducibly displayed an irreversible rise of p53, multi-drug resistant emergence may not be a suitable cancer treatment option. Patients treated with MDM2 inhibitor such as Nutlin-3 should be monitored for the rise of p53 mutations in multi-drug resistant cells (Michaelis). Intrinsic resistance is pre-existent and the occurrence is present prior to receiving treatment, meaning resistance mutations can be present in a small subpopulation of tumour cells before the initiation of therapy (Foo). This is usually a result of the acquisition of stochastic mutations (Zahreddine). The emerging resistance mutations would be fixed within the population of tumour cells, after the initiation of treatment the sub-population of cells carrying the mutation would increase and survive the insult of therapy (Friedman). With intrinsic resistance, malignant cells exposed to aggressive treatments will have an increased likelihood of their pre-existing mutations dictating the tumour cell population. Patients would then fail to respond with initial treatment (Housman). Combination therapies are currently being investigated in clinical research, as a mechanism to overcome resistance to molecularly targeted therapies. Current studies have demonstrated that the addition of CDK4/6 inhibitors in combination with existing therapies can potentially improve patient response and overcome treatment resistance (Hamilton). The cell cycle is highly regulated by cyclin-dependent kinases (CDKs), but dysregulation of CDK activity had been detected in a broad range of malignancies (Hamilton). Aberrant CDK activity can result in continuous growth and abnormal cell cycle proliferation, and underlying causes can include: gene amplification or rearrangement, loss of negative regulators, epigenetic alterations, and point mutations in key pathway components (Hamilton). Existing treatments that are being investigated for potential combination therapy with CDK4/6 inhibitors include: hormonal therapy, PI3K/AKT/mTORpathway inhibitors, RAS/RAF/MEK/ERK pathway inhibotrs, chemotherapy, and radiotherapy (Hamilton). Preclinical studies have shown promising results in combination therapies. Abemaciclib had a synerginistic effect on chemotherapy agent gemicitabine (Gelbert), and palbociclib diminished cytotoxic effects of antimitotics and platinum agents (Gogolin). Inhibitors of cyclin-dependent kinases (CDKs) 4/6 have shown efficacy and clinical activity in several malignancies (Knudsen). Targeting the cell cycle with a powerful class of novel agents will strike a key characteristic in cancer (Knudsen). Due to the rise of resistance limiting the effectiveness of chemotherapy and cytotoxic drugs, researchers are investigating personalised medicine in treating cancer. Using the systemic use of genetic information can be beneficial in order understand the molecular basis of cancer (Garraway, Mukesh Verma). Personalised medicine involves utilizing this information based on a patient’s individual genetic makeup, and it will provide a genetic understanding of their disease, in order to tailor therapeutic care to that patient’s needs (Mukesh Verma). This has been demonstrated with a non-invasive analysis of circulating tumour DNA (ctDNA). As previously mentioned, patients with advanced cancer undergo prolonged systemic treatments are likely to acquire resistant due to clonal evolution (Murtaza). Routinely, serial tumour biopsies are conducted to further analyse the genomic modification caused by the treatment, are usually invasive and can be misperceived by tumour heterogeneity (Murtaza). Previous studies have demonstrated that ctDNA is more accessible, easier to process, and contains representation of the entire tumour genome (Murtaza) The ctDNA would also contain mixed variants that had originated from a variety of independent tumours (Murtaza). In this preliminary study, six patients that had the following advanced malignancies: breast cancer, ovarian cancer, or lung cancers were followed over 1-2 years. Exome sequencing on a variety of samples with each case, and showed proof of genomic evolution and displayed genome-wide similarities between tumour DNA and ctDNA (Murtaza). The mutations discovered were found to either be well-recognized oncogenic genes that have been associated with drug resistance and drug metabolism, or they were newly discovered genes that had not been known to be linked to drug resistance or carcinogenesis (Murtaza). One of the patients in the study, with advanced ovarian cancer was treated with cisplatin. After the treatment, she had an abundance of mutations in the tumour-suppressor RB1 gene, which is known for inactivating the RB1 protein (Murtaza). Her matched metastasis biopsy revealed the mutation was found in 95% of sequencing reads, with a loss of heterozygosity at 13q containing the RB1 gene (Murtaza). Loss of this gene had also been associated with chemotherapy response (Murtaza). Other studies have used this non-invasive technique to analyse ctDNA in EGFR-TKI mutations, and various Lung Cancers (Zhang, Fiala).