An investigation of the role of the hypersialylated myeloma cell surface in facilitating evasion of natural killer cell-mediated immunosurveillance
Daly, John
Daly, John
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Publication Date
2021-12-30
Type
Thesis
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Abstract
Abnormal glycosylation is a hallmark of cancer, and hypersialylation in particular has been shown to facilitate abnormal cell trafficking and drug resistance in many cancers, including Multiple Myeloma (MM) - a currently incurable plasma cell malignancy. Hypersialylation has also been implicated in facilitating evasion of natural killer (NK) cell-mediated immunosurveillance by tumour cells. NK cells are cytotoxic lymphocytes with an innate ability to recognize and destroy virally infected, genetically damaged and malignantly transformed cells and are vital effectors of the innate immune response. Further studies have implicated sialic acid-binding inhibitory NK cell receptors, termed Siglecs, in regulating NK cell functions against Siglec ligand-expressing tumour cells. While advancements in MM therapy have ensured patient outlook has improved considerably in recent decades, the majority of MM patients entering remission will inevitably relapse with more significant health maladies. Therefore, it is imperative to investigate potential mechanisms of resistance utilised by MM cells to evade immunosurveillance by the patient’s immune system. In this study the role of hypersialylation in facilitating immune-escape from NK cells in MM was investigated, with a focus on generating novel NK cell-based therapies to enhance anti-MM functions. While hypersialylation has been implicated in facilitating evasion of NK cell-mediated immunosurveillance by several cancers, prior to this project there had been no investigations into this axis within MM. Therefore, the first phase of this project was to complement existing research in other cancer types by determining whether desialylation of the MM cell surface could enhance NK cell cytotoxicity. Further initial exploratory assays were used to determine the expression of Siglec ligands, and their cognate receptors, on MM cells and NK cells respectively, to confirm whether the Siglec-Siglec ligand axis could potentially be hijacked by MM cells to dampen NK cell responses. Upon MM cell surface desialylation strongly enhanced NK cell-mediated cytotoxicity was observed against several classical MM cell lines, confirming that cell surface hypersialylation protects MM cells from NK cell-mediated immunosurveillance. Screening of primary MM cells, isolated from patient bone marrow aspirates, and a panel of MM cell lines revealed strong expression of ligands for both Siglec-7 and Siglec-9. Furthermore, analysis of primary NK cells; either resting, activated or expanded, sourced from both healthy donors and NK cells from BMAs of MM patients revealed high expression of Siglec-7. Siglec-7 was expressed at higher levels in NK cells from MM patient BMAs than in NK cells from the peripheral blood of healthy donors, confirming the potential of Siglec-7/Siglec-7 ligand interactions within the MM tumor microenvironment to be exaggerated and therefore contribute to suppression of NK cell anti-cancer functions. Siglec-9 expression was also observed on subsets of naïve and IL-2 activated primary NK cells, but was absent on expanded NK cells and NK cell lines, presenting Siglec-7 as a promising candidate for further investigations. Finally, mass spectrometry of Siglec-7 Fc chimera-binding proteins after incubation with MM cell line lysates revealed PSGL-1 and CD43 as ligands for Siglec-7, with PSGL-1 more widely expressed than CD43. Elucidating the identity of these ligands presents PSGL-1 and CD43 as novel target antigens which can be targeted to abolish Siglec-7-mediated NK cell regulation against MM cells. Hypersialylation of the tumour cell surface has been hypothesized to physically mask activating ligands which could otherwise promote NK cell anti-cancer functions. Desialylation of MM cells has previously been shown to increase detection levels of BCMA, an antigen targeted by monoclonal antibody therapy to stimulate NK cell-mediated ADCC. Upon desialylation of MM cell lines and screening for several different target antigens, increased detection of the glycoprotein CD38 was observed. CD38 is a target of anti-CD38 monoclonal antibodies used in the clinic to treat MM, such as Daratumumab (Dara). Following desialylation MM cells exposed to Dara were sensitized to NK cell-mediated ADCC compared to Dara treatment alone. To address MM cells with low CD38 expression, which can be observed following Dara treatment in MM patients, MM cells were treated with ATRA to upregulate CD38 expression. Combining ATRA treatment with SIA-mediated desialylation and Dara elicited a potent NK cell-mediated cytotoxic response, which was significantly stronger than individual treatments of the same MM cells. Finally, CD38 KO in NK cells using CRISPR/Cas9 was proposed as a means of potentially preventing fratricide, a limiting factor of therapeutical response observed in moAb-based immunotherapies heavily dependent on NK cell-mediated responses, such as Dara. The final phase of this project was to investigate the potential of targeting promising NK cell immune checkpoints such as Siglec-7 and CD96 to enhance NK cell anti-MM cytotoxicity. Having identified Siglec-7 as a promising novel checkpoint inhibitor in regulating NK cell cytotoxicity against MM and, using CRISPR/Cas9 to genetically delete Siglec-7 expression, Siglec-7KO NK cells displayed enhanced cytotoxicity against Siglec-7L+ MM cell lines, confirming that MM cells can evade NK cell-mediated immunosurveillance in a Siglec-7-dependent manner. However, it is not yet apparent whether enhanced NK cell-mediated cytotoxicity observed upon MM cell surface desialylation is entirely as a result of abolished Siglec-7-Siglec-7 ligand interactions, and may potentially be caused, at least in part, by exposing ligands for activating NK cell receptors previously masked by sialic acids. Further CRISPR/Cas9-based studies in this chapter targeted CD96 - an NK cell expressed receptor with an unclear role in human NK cells with contradictory data suggesting CD96 to be both an inhibitory and activating NK cell receptor. Cytotoxicity and cytokine release assays revealed conclusively that CD96 is an inhibitory NK cell receptor, with CD96KO NK cells mediating higher cytotoxicity and cytokine release against CD155+ MM cell lines, compared to CD96+ NK cells from the same donor. While blocking antibodies have been used to target NK cell immune checkpoints, highly efficient targeting of immune checkpoints using CRISPR/Cas9 MM has not previously demonstrated. Thus, this phase of the project highlights Siglec-7 and CD96 as important NK cell immune checkpoints, as well as proposing CRISPR/Cas9 as a promising tool to engineer potent NK cells lacking inhibitory pathways that can be hijacked by cancer. Taken together, this project demonstrates several novel mechanisms of enhancing NK cell activity against MM. Targeted desialylation of the hypersialylated MM cell surface is a promising strategy to enhance NK cell efficacy against MM, which can be combined with existing frontline therapies, such as anti-CD38 monoclonal antibodies, to elicit a potent anti-MM response by combing enhanced cytotoxicity through abolished Siglec-7 inhibitory signalling while simultaneously enhancing ADCC. Finally, CRISPR/Cas9 represents a novel mechanism for generating highly potent NK cells, potentially suitable as part of adoptive NK cell-based therapies which could deliver more enhanced anti-MM responses.
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NUI Galway