Exploring the role of STING type-I Interferon response as a therapeutic in myeloid leukaemia

Abstract

Myeloid leukaemia is a type of blood cancer that originates from the development of abnormal myeloid cells in the bone marrow and classified into either acute or chronic myeloid leukaemia (AML/CML). Acute Myeloid Leukaemia (AML) poses a unique challenge in haematological oncology due to its aggressive nature and significant genetic diversity. Unlike Chronic Myeloid Leukaemia (CML), which is primarily driven by the BCR-ABL1 fusion gene and can be effectively managed with tyrosine kinase inhibitors (TKIs). Intensive chemotherapy is the front-line treatment for AML, and not all patients fit or can tolerate it owing to its toxicity. Targeted therapies customised to the molecular and cytogenetic profiles of each patient have been administered for AML treatment and improved the management of AML. Nevertheless, their efficacy is often limited, since some individuals may acquire resistance over time. Thus, AML patients have the worse overall survival (OS) compared to CML patients. Type-I IFN signalling through STING pathway appears to negatively impact the development of leukaemia, in line with its downstream functions, such as increase in rate of apoptosis, increase in AML immunogenicity, and decrease in cell proliferation. STING is found highly expressed in AML, making it an appealing therapeutic target. Its activation has the potential to overcome the immunosuppressive bone marrow microenvironment, where conventional immune responses are less effective, and could stimulate a robust immune response against AML. This highlights the promise of STING-based therapies in a disease where conventional treatments often fall short, offering a new strategy to address the challenges posed by AML's genetic complexity. The role of STING gene in the outcome of AML were studied through various approaches including bioinformatics, cellular and molecular biology and immunoassay techniques. Bioinformatics databases were used to analyse and evaluate STING expression in AML and how this affects the OS and patients’ prognosis. The efficacy of STING agonist (CDA) in human AML and CML cell lines was assessed by western blotting to study the functioning of STING signalling pathway in AML and CML, and RT-qPCR to evaluate the production of type-I IFN and its ability to induce of innate immune response in those cells. Flow cytometry was used to assess apoptosis and cell proliferation. Application of Immune assay in a co-culture system enabled us to study tumour microenvironment in vitro by assessing immune cells response against AML/CML upon STING activation in the co-culture through performing T-cell proliferation, NK cell lysis of tumour cells (ADCC assay), macrophage phagocytosis (ADCP assay) and Dendritic cells efferocytosis assay. Finally, Studying the synergetic effect of STING agonist in combination with hypomethylating agent such as 5’Azacytidine and how these combinations enhance tumour anti-immunity. Publicly available database was used for bioinformatic analysis and showed that STING gene is highly expressed and associated with myeloid lineage while MAVS gene which encodes Mitochondrial Antiviral Signalling Protein (MAVS) was associated with lymphoid lineage in HSCs. Significantly, elevated STING expression was positively correlated with increased overall survival in AML dataset (OHSU, TCGA). An In vitro study demonstrated that STING agonist was able to activate STING pathway in AML and CML cell lines within 1 hour, leading to IFN-β upregulation. I demonstrated that STING pathway activation inhibited proliferation and induced apoptosis. These physiological changes upon STING activation were accompanied by alterations in the AML and CML immunophenotype within 24 hours such as (HLA A,B,C and CD86). Furthermore, the In vitro study of the tumour microenvironment through employing immune cells and AML/CML cells in a co-culture system showed that treating the co-culture with STING agonist restores CD8 T-cell proliferation comparing to suppressed CD8 T-cells in untreated co-culture. STING agonist also enhanced macrophage and dendritic cells phagocytosis in the co-culture to the target cells when measured by flow cytometry and upregulated the activator FcγR and reduced the inhibitory one in macrophage as well as enhanced antigen presenting markers on DC. Significantly, transcriptomic analysis revealed that the high state of immune response induced by a hypomethylating agent treatment 5-Azacytidine (5'Aza), through "viral mimicry induction," enhanced the sensitivity of an AML cell line that was less responsive to STING activation when treated with the STING agonist alone, making the cell line more responsive to combination therapy. Taken together, these results pointed to the synergetic effect of STING agonist in combination with chemotherapy such as 5’Aza and these combinations enhanced tumour anti immunity.