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Biological Evaluation of Novel Kinase Inhibitors against ALK, FGFR, c-KIT and BCR-ABL for Overcoming Drug Resistance

  • Yunju Nam (남윤주, KU-KIST융합대학원 NBIT융합전공)

초록/요약

Kinase is one of the enzymes that plays a key role in regulation the cellular signaling systems. There are more than 500 protein kinases and they are specifically involved in cell growth, proliferation and differentiation. Important mutations of kinases result in abnormal proliferation, and such uncontrolled growth and metastasis is major characteristic of cancer cells. By 2020, 68 kinase inhibitors targeting 24 different protein kinases have been approved, and they are attracting attention as important therapeutic agents for various diseases, including cancer, immune diseases, neurological diseases and metabolic diseases. However, despite the development of kinase inhibitors, there is a limitation in acquiring drug resistance through a target kinase mutation. For this reason, one of the key considerations in the development kinase inhibitors is various attempts to overcome drug resistance. In this thesis, we evaluated and explored novel kinase inhibitors targeting ALK, FGFR, c-KIT and BCR-ABL which are major kinases for cancer therapy. By establishment of stable cells with resistant mutants of kinases, we were able to discover new kinase inhibitors that can inhibit resistant mutations in cellular context. In part 1, from results of structure-activity relationship (SAR) using establishment of mtALK-TEL Ba/F3 cell lines showing drug resistance, we discovered Anaplastic Lymphoma Kinase (ALK) inhibitors that can potently inhibit the clinically relevant mutants of ALK kinase. The discovered inhibitors significantly suppressed proliferation of cancer cells harboring ALK mutations via inhibition of ALK signaling, cell cycle arrest, and apoptosis induction. In part 2, we showed that GNF-7, a multi-targeted kinase inhibitor, and its derivatives including SIJ1263 (IC50 < 1 nM against FGFR4) are highly potent FGFR4 inhibitors. Hepatocellular carcinoma (HCC) is disease with a high mortality rate and limited treatment options. Alterations of fibroblast growth factor receptor 4 (FGFR4) has been regarded as an oncogenic driver for HCC and a promising target for HCC therapeutics.GNF-7 and SIJ1263 are capable of strongly suppressing proliferation of HCC cells and Ba/F3 cells transformed with wtFGFR4 or mtFGFR4. Compared with known FGFR4 inhibitors, both GNF-7 and SIJ1263 possess much higher (up to 100-fold) anti-proliferative activities via FGFR signaling blockade and apoptosis on HCC cells. Especially, SIJ1263 is 80-fold more potent on TEL-FGFR4 V550E Ba/F3 cells than BLU9931, which suggests that SIJ1263 would be effective for overriding drug resistance. In addition, both substances strongly suppress migration/invasion and colony formation of HCC cells. In part 3, new derivatives capable of inhibiting FGFR mutations, including gatekeeper mutations were studied. Although FGFR inhibitors hold promise in treating various cancers, resistance to the FGFR inhibitors caused by acquired secondary mutations has emerged. As a result of the SAR study, 3-16a was identified as a very potent panFGFR inhibitor against wild-type and mutant FGFR. Notably, 3-16a is superior to the approved FGFR inhibitor infigritinib against V555M-FGFR3. In addition, 3-16a and analog 3-17a inhibit proliferation of cancer cells harboring FGFR mutations through blockade of FGFR signaling, cell cycle arrest, and apoptosis. In addition, 3-17b significantly reduced tumor growth in the AN3CA mouse xenograft model. This study provides new insights into the design of novel FGFR inhibitors that are active against FGFR mutations. In part 4, the SAR studies led to the identification of the derivative 4-1r as a potent c-KIT inhibitor. c-KIT has been regarded as a promising therapeutic target against gastrointestinal stromal tumor (GIST). Overcoming drug resistance of c-KIT inhibitors including imatinib is required. We performed structure-activity relationship (SAR) studies to overcome imatinib resistance. The derivative 4-1r is capable of strongly inhibiting a c-KIT V560G/D816V double mutant that is resistant to imatinib and remarkably attenuates proliferation of GIST-T1 and HMC1.2 cancer cells. Moreover, 4-1r possesses differential cytotoxicity on c-KIT D816V Ba/F3 cells relative to parental Ba/F3 cells. Kinase panel profiling revealed that 4-1r has reasonable kinase selectivity. Furthermore, 4-1r not only blocks migration and invasion, but also suppresses anchorage-independent growth of GIST-T1 cells. In part 5, we showed that novel derivatives as highly potent and selective on BCR-ABL-T315I with marginal VEGFR2 inhibition. Tremendous efforts devoted to overriding the imatinib resistance mainly derived from ABL kinase domain point mutations. Although ponatinib is the only clinically approved inhibitor to overcomes T315I BCR-ABL gatekeeper mutant, it exhibits lethal side effects such as cardiac toxicity due to its significant VEGFR2 inhibitory activity. The optimized derivative 5d is a highly selective kinase inhibitor and exhibits 40-fold more potent activity at T315I BCR-ABL than VEGFR2. 5d strongly suppresses proliferation of BCR-ABL-T315I Ba/F3 cells via apoptosis and BCR-ABL signaling blockade. Furthermore, 5d displayed significant in vivo efficacy in bioluminescent xenograft models using T315I BCR-ABL Ba/F3 cell lines without notable adverse effects. In part 6, We investigated whether the multi-targeted kinase inhibitor, GNF-7, could effectively inhibit M2 polarization of macrophages. Reprogramming of M2 polarized tumor-associated macrophages (TAMs) is one of the important therapeutic goals in cancer treatment. GNF-7 reduced the distribution of CD206 as M2 marker and the expression level of Arg-1 in both RAW264.7 cells and bone marrow-derived macrophages (BMDMs). In addition, the expression level of specific M1-marker gene, iNOS was enhanced by GNF-7, and we identified GNF-7 as a modulator of M2 polarized TAM. Among the kinases inhibited by GNF-7, Src kinase is associated with the reprogramming of M2 polarized TAMs. Moreover, the treatment of GNF-7 enhances the immune-stimulatory cytokine TNF-α and induces the apoptosis of HT29 and B16 cells toward an inflammatory phenotype. The effect of GNF-7 was dependent on signaling pathways including Src kinase. Taken together, GNF-7 suppresses cancer progression by regulating macrophage polarization.

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목차

Contents i
Abstract viii
General Introduction xii
References xiv
List of Figures xviii
List of Tables xxi
List of Abbreviations xxiii
Chapter 1. Biological evaluation of aminopyrazolopyridine derivatives as ALK inhibitors for overcoming drug resistance 1
1.1 Introduction 1
1.2 Materials and Methods 4
1.2.1 Chemical synthesis 4
1.2.2 Establishment of ALK mutant transformed Ba/F3 cells 4
1.2.3 Cell culture and reagents 6
1.2.4 Anti-proliferation assay 6
1.2.5 Western blot 6
1.2.6 Apoptosis analysis and cell cycle arrest 7
1.3 Results 8
1.3.1 Structure activity relationship 8
1.3.2 Kinase profile of 1-1g 12
1.3.3 Inhibition by 1-1g of ALK signalling 15
1.3.4 Effects of 1-1g on apoptosis and cell cycle arrest 17
1.3.5 Further optimization of derivatives and SAR study 20
1.3.6 Effects of 1-4g in L1196M/G1202R ALK-TEL Ba/F3 cells 27
1.4 Discussion 29
1.5 References 31
Chapter 2. Anti-cancer effects of GNF-7 and its derivatives on hepatocellular carcinoma harboring FGFR4 activation 36
2.1 Introduction 36
2.2 Materials and Methods 38
2.2.1 Chemical synthesis 38
2.2.2 Cell culture and reagents 38
2.2.3 Biochemical in vitro kinase assay and kinome profiling 38
2.2.4 Cell proliferation assay 38
2.2.5 RT-PCR 39
2.2.6 Cell lysis and western blotting 39
2.2.7 Cell cycle arrest 39
2.2.8 Apoptosis assay 40
2.2.9 Migration assay 40
2.2.10 Invasion assay 40
2.2.11 Soft agar assay 40
2.3 Results 42
2.3.1 Enzymatic activities of GNF-7 against FGFR1-4 and their mutants 42
2.3.2 Anti-proliferative activities of GNF-7 44
2.3.3 SAR study and establishment of ETV6-FGFR4 Ba/F3 cells 46
2.3.4 Molecular docking studies of GNF-7 and SIJ1263 on wtFGFR4 and FGFR4 V550E 53
2.3.5 Kinome-wide inhibition profiling of SIJ1263 53
2.3.6 Anti-proliferative activities of GNF-7 and SIJ1263 55
2.3.7 Effects of GNF-7 and SIJ1263 on FGFR signaling 59
2.3.8 GNF-7 and SIJ1263 induce apoptosis and cell cycle arrest in HCC cells 61
2.3.9 GNF-7 and SIJ1263 suppress migration and invasion of HCC cells 64
2.3.10 Suppression of anchorage-independent growth by GNF-7 and SIJ1263 67
2.4 Discussion 69
2.5 References 71
Chapter 3. Biological evaluation of pyridinyltriazine derivatives as potent panFGFR inhibitors against gatekeeper mutants for overcoming drug resistance 76
3.1 Introduction 76
3.2 Materials and Methods 78
3.2.1 Chemical synthesis 78
3.2.2 Cell culture 78
3.2.3 Cell viability assays 78
3.2.4 Western blot analysis 78
3.2.5 Apoptosis assay 79
3.2.6 Migration assay 79
3.2.7 Invasion assay 79
3.2.8 Soft agar assay 79
3.2.9 In vivo efficacy study 80
3.2.10 Immunohistochemistry 80
3.2.11 Statistical analysis 80
3.3 Results 81
3.3.1 Enzymatic inhibitory activities of 3-1, 3-9, and 3-16a 81
3.3.2 SAR study and establishment of ETV6-FGFR3 V555M Ba/F3 cells 83
3.3.3 Kinome-wide profiling 92
3.3.4 Effects on FGFR signaling 94
3.3.5 Effects on apoptosis induction and cell cycle arrest 96
3.3.6 Suppression of migration and invasion 99
3.3.7 Suppression of anchorage-independent growth 102
3.3.9 in vivo efficacy 104
3.3.11 Exploring a new indication of 3-17a 108
3.4 Discussion 111
3.5 References 113
Chapter 4. Biological evaluation of thiazolopyridine derivatives as c-kit inhibitors for overcoming imatinib resistance 118
4.1 Introduction 118
4.2 Materials and Methods 120
4.2.1 Chemical synthesis 120
4.2.2 Cell culture and reagent 120
4.2.3 In vitro kinase assay 120
4.2.4 Cell viability assay 120
4.2.5 Western blot analysis 121
4.2.6 Cell cycle arrest and apoptosis analysis 121
4.2.7 Migration assay 122
4.2.8 Invasion assay 122
4.2.9 Soft agar assay 122
4.3 Results 123
4.3.1 Cancer cell profiling 123
4.3.2 SAR study 125
4.3.3 Effects on c-KIT signaling 130
4.3.4 Effects on apoptosis and cell cycle arrest 132
4.3.5 Effects on cell migration and invasion 135
4.3.6 Effects on anchorage-independent growth 137
4.4 Discussion 139
4.5 References 141
Chapter 5. Biological assessment of selective Type II BCR/ABL-T315I inhibitors with reduced adverse effects 143
5.1 Introduction 143
5.2 Materials and Methods 145
5.2.1 Cell Culture and reagent 145
5.2.2 Anti-proliferation assay 145
5.2.3 Western blot 145
5.2.4 Apoptosis analysis 146
5.2.5 in vivo efficacy 146
5.3 Results 147
5.3.1 SAR study 147
5.3.2 Effects on BCR/ABL signaling 149
5.3.3 Effects on apoptosis induction 151
5.3.4 in vivo efficacy 153
5.4 Discussion 155
5.5 Reference 157
Chapter 6. GNF7 reprograms M2 polarization of tumor-associated macrophages 161
6.1 Introduction 161
6.2 Materials and Methods 163
6.2.1 Cell culture and polarization of macrophages 163
6.2.2 Cell proliferation assay 163
6.2.3 Co-culture methods 163
6.2.4 RT-PCR 163
6.2.5 Cell cycle arrest 164
6.2.6 Apoptosis assay 164
6.2.7 Migration assay 164
6.2.8 Cell lysis and western blotting 164
6.2.9 BMDMs isolation 165
6.2.10 Cytokine assay 165
6.2.11 Statistical analysis 165
6.3 Results 166
6.3.1 GNF7 efficiently inhibits M2 polarization of TAMs 166
6.3.2 Exploring the pathways inhibited by GNF-7 in TAM polarization 169
6.3.3 GNF-7 increases anti-cancer effects by reprogramming of macrophages 173
6.4 Discussion 175
6.5 Reference 177
Abstract in Korean 183

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