01whole.pdf (4.73 MB)
Examining the response of melanoma cells to immune activity and immunotherapy
thesisposted on 2022-03-28, 23:58 authored by Sara Alavi
Immunotherapy in the form of immune checkpoint inhibitors has significantly improved the survival of patients with advanced melanoma. However, immune checkpoint blockade is only effective in 15-40% of melanoma patients and failure to respond to immune checkpoint blockade may occur via several tumour intrinsic or extrinsic mechanisms, including overexpression of immune inhibitory ligands and receptors, alterations in antigen processing and presentation, and defects in the interferon gamma (IFNγ) signalling pathway. In this PhD project, we first examined IFNγ responses in a large panel of immunotherapy-naïve melanoma cell lines with defined genetic drivers; BRAF-mutant, NRAS-mutant, BRAF/NRAS wild type cutaneous melanoma, and GNAQ/GNA11-mutant uveal melanomas (Chapter 2). We investigated the basal and IFNγ-induced expression of immune inhibitory ligands PD-L1 and PD-L2, antigen presenting molecules HLA-ABC and HLA-DR, and nerve growth factor receptor (NGFR) on the surface of these immunotherapy-naïve cell lines to determine the influence of melanoma driver oncogenes on IFNγ signalling. In Chapter 3, we compared tumour necrosis factor (TNFα) response in the same panel of cell lines. We found that melanoma response to IFNγ and TNFα are heterogeneous, and that IFNγ induced PD-L1, PD-L2, HLA-DR and HLA-ABC expression more potently, whereas TNFα preferentially up regulated NGFR expression. We further identified two well-recognised mechanisms of immunotherapy resistance, including the loss of β-2-microglobulin (β2M) and interferon gamma receptor 1 (IFNGR1) expression in these immunotherapy-naïve cells. In Chapter 4, we extended our characterization of resistance mechanisms to a panel of 16 short-term melanoma cell lines (PD-1 PROG cell lines) derived from patients who progressed on anti-PD-1 or combination of anti-PD-1 and anti-CTLA-4 based immunotherapy. We assessed expression of IFNGR1, PD-L1, PD-L2, HLA-ABC, HLA-DR and B2M in these cells. We additionally analysed expression of transcription factors, melanoma pigment antigens and markers of de-differentiation, including SOX10, MLANA, AXL, MITF and NGFR. We identified several potential mechanisms of immunotherapy resistance in this panel of PD-1 PROG cell lines including loss of β2M expression, increased expression of immune inhibitory molecules, diminished response to IFNγ stimulation and melanoma de-differentiation. Finally, in Chapter 5, we examined the regulation of PD-L1 and PD-L2 in the melanoma cell lines. Specifically, we assessed the temporal accumulation and stability of total and cell surface-specific expression of PD-L1 and PD-L2. Our results demonstrate that melanoma responses to IFNγ or TNFα are heterogeneous, frequently downregulated in immunotherapy-naïve melanoma and potentially predictive of response to immunotherapy. Our results also confirmed that loss of antigen presentation molecules, upregulation of immune inhibitory checkpoints, loss of melanoma response to IFNγ and reduced expression of melanoma differentiation antigens, are associated with resistance to PD1-based immunotherapies. Translation of these findings to the clinic may provide clinicians with the rational design and clinical development of personalized treatment strategies for each patient, which could result in better treatment response and increased long-term survival rates for patients with metastatic melanoma.