Genomic Determinants of Drug Sensitivity in Colorectal Cancer : PIK3CA Mutations Predict Resistance to PIM1 Inhibitors and TP53/DNA-PK Alterations Predict Sensitivity to Olaparib

다국어 초록 (Multilingual Abstract)

Colorectal cancer (CRC) exhibits substantial molecular heterogeneity, leading to highly variable responses to targeted therapies among patients. To address this clinical variability and enable personalized treatment, the identification and application of predictive biomarkers are essential. Predictive biomarkers provide molecular indicators that can anticipate tumor sensitivity or resistance to specific drugs. Their use can reduce unnecessary treatment, shorten therapy duration and costs, minimize side effects, and improve overall treatment efficiency. This thesis aims to investigate the genomic determinants of drug sensitivity in CRC and to establish patient-tailored targeted therapy strategies.
First, PART 1 examines PIK3CA mutations, which encode the p110α subunit of Class I phosphoinositide 3-kinase (PI3K), as potential negative predictive biomarkers for PIM1 (proviral integration site for Moloney murine leukemia virus 1) inhibitors. Previous studies have shown that PIK3CA mutations confer relative resistance to PIM1 inhibitors in prostate and non-small-cell lung cancers, likely due to persistent activation of the PI3K–AKT–mTOR signaling cascade that functionally overlaps with PIM kinase signaling. However, this phenomenon has not yet been systematically evaluated in CRC, and it remains unclear whether PIK3CA mutations elicit similar resistance in this tumor context. Therefore, assessing PIK3CA status alongside the response to PIM inhibitors in CRC may help establish PIK3CA as a predictive biomarker for guiding PIM1-targeted therapy.
PART 2 investigates whether alterations in TP53 and DNA-dependent protein kinase (DNA-PK) can serve as predictive biomarkers for response to the PARP inhibitor olaparib. Although olaparib is known to be effective primarily in tumors with BRCA1/2 mutations, CRC represents an exception in which therapeutic outcomes appear to be influenced by TP53 genotype. Previous research has demonstrated that TP53–wild-type CRC cell lines are sensitive to olaparib, whereas TP53-mutant lines exhibit resistance, with several mechanisms proposed to explain this differential response. Given that DNA-PK is one of the major regulators of p53 activation, this study further explores whether DNA-PK alterations enhance olaparib efficacy. By evaluating and validating the impact of DNA-PK alterations on olaparib response, PART 2 aims to identify novel predictive biomarkers that could refine patient selection and improve the clinical applicability of PARP inhibition in CRC.
This thesis highlights the value of genomic status of specific genes for predictive biomarker discovery in CRC. By identifying PIK3CA mutation mediated resistance to PIM1 inhibitors and TP53/DNA-PK–dependent sensitivity to olaparib, it provides a foundation for patient-tailored therapies that may enhance efficacy, reduce unnecessary treatments, and optimize clinical outcomes.

목차 (Table of Contents)

• Contents i
• General Abstract v
• PART 1 1
• List of Abbreviation 2
• List of Figures 3
• Abstract 5
• 1. Introduction 7
• 1.1. Current therapeutic approach in colorectal cancer treatment 7
• 1.2. Role of PI3Ks in cell growth and survival 7
• 1.3. PI3K mutations in cancer 8
• 1.4. Function of PIM (proviral integration site for Moloney murine leukemia virus) kinases in normal and cancer 9
• 1.5. Aim of current study 10
• 2. Material and Methods 12
• 2.1. Cell culture and reagents 12
• 2.2. DNA constructs and transfection 12
• 2.3. Trypan blue cell exclusion assay (cell death assay) 12
• 2.4. In vitro colony-forming assay 12
• 2.5. Annexin V-FITC/PI staining (apoptosis assay) 13
• 2.6. Sample preparation and Western blot analysis 13
• 2.7. In vivo xenograft and Patient Derived Xenograft (PDX) models 13
• 2.8. Statistical analysis 14
• 3. Results 15
• 3.1. Identification of PIK3CA mutation as the underlying factor for resistance to SMI-4a in various CRC cell lines 15
• 3.2. PIK3CA mutation induces primary resistance to SMI-4a treatment in CRC cell lines 17
• 3.3. Overexpression of PIK3CA mutants shows resistance to SMI-4a treatment 21
• 3.4. Differences in response to SMI-4a in tumor xenografts, depending on PIK3CA genotype 23
• 3.5. Co-treatment of NVP-BKM120 in PIK3CA mutated CRC cell lines restores SMI-4a sensitivity 27
• 4. Discussion 31
• References 35
• PART 2 42
• List of Abbreviation 43
• List of Figures 44
• Abstract 46
• 1. Introduction 47
• 1.1. DNA Damager Response (DDR) in cancer 47
• 1.2. The Poly ADP Ribose Polymerase (PARP) and PARP inhibtors (PARPi) 48
• 1.3. DNA-dependent protein kinase (DNA-PK) and DDR 49
• 1.4. Colorectal cancer (CRC) and PARPi 50
• 1.5. p53 and cancer therapy 50
• 1.6. Combination strategy with p53 and PARPi 51
• 1.7. Aim of current study 52
• 2. Material and Methods 53
• 2.1. Cell culture and generation of stable cell lines 53
• 2.2. Cell transfection with shRNA 53
• 2.3. Plasmids and transfections 53
• 2.4. Trypan blue cell exclusion assay (cell death assay) 54
• 2.5. Cell viability assay 54
• 2.6. Flow cytometry analysis 54
• 2.7. Western blot analysis 55
• 2.8. Immunofluorescence 55
• 2.9. Caspase-3/7 activation analysis 56
• 2.10. Chromosome aberration analysis 56
• 2.11. In vivo xenograft and Patient Derived Xenograft (PDX) model 56
• 2.12. Patients and specimens 57
• 2.13 Reverse transcription-polymerase chain reaction (RT-PCR)· 57
• 2.14. Immunohistochemistry 57
• 2.15. Statistical analysis 58
• 3. Results 59
• 3.1. The sensitivity of colorectal cancer cells to Olaparib varies depending on their TP53 and DNA-PK genotypes 59
• 3.2. DNA damage response induced by Olaparib is differentiated with respect to p53 and DNA-PK genotypes 67
• 3.3. The anticancer effect of Olaparib in CRC cells is determined by p53 and DNA-PK genotypes 72
• 3.4. Loss of DNA-PK affects Olaparib-induced genomic instability and NHEJ repair in p53 WT cells 79
• 3.5. The anti-cancer effects of Olaparib in patient-derived cancer cells (PDCs) and patient-derived xenograft (PDX) models are mediated by a DNA-PK and p53-dependent mechanism 82
• 4. Discussion 89
• References 91
• Abstract in Korean 97
• Publications 101