Investigating the usage of the histone deacetylase inhibitor entinostat and novel c-FLIP inhibitors for the prolongation of transplant survival via induction of T cell apoptosis

Abstract

Organ transplantation remains the key, and only long term, treatment option for end stage organ failure. The development of immunosuppressants capable of inhibiting episodes of acute rejection, which had limited the success of early transplantation efforts, has allowed for successful organ transplantation with the ability to treat acute rejection. However, there has been a dearth of novel immunosuppressive agents for use within transplantation over the last two decades. Further, current immunosuppressive agents broadly suppress the immune system, leaving the patient susceptible to malignancy and infection. Current immunosuppressant agents have limited effectiveness with regards to preventing the loss of the transplanted organ to chronic rejection. Thus, the development of novel immunosuppressive agents capable of selectively targeting the alloreactive immune cells which drive rejection, whilst leaving the resting cells untouched, would prove immensely beneficial, preventing graft rejection whilst possessing fewer side effects than current immunosuppressants.

Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP) is an anti-apoptotic protein, which in newly stimulated T cells prevents apoptosis. In the oncology setting, c-FLIP has been shown to be associated with a poorer prognosis, as it renders malignant cells resistant to apoptosis. Therefore, the inhibition of c-FLIP has been explored as a possible therapeutic option for cancer. Histone deacetylase inhibitors (HDACi) and novel small molecule c-FLIP inhibitors (c-FLIPi) have been found to inhibit c-FLIP activity resulting in apoptosis of cancerous cells. The aim of the studies outlined in this thesis was to investigate the potential of repurposing HDACi and c-FLIPi to inhibit c-FLIP, induce apoptosis of alloreactive T cells which drive rejection, and consequently prolong graft survival.

It was hypothesised that 1) HDACi and c-FLIPi induce apoptosis and prevent proliferation of activated T cells, 2) that this apoptosis would be driven by caspase-8, and 3) that the use of HDACi and c-FLIPi in a model of transplantation results in prolonged graft survival, as a result of apoptosis within alloreactive T cells capable of driving rejection. To test these hypotheses; firstly, the effect of HDACi and c-FLIPi was examined using in vitro models of activated murine and human peripheral blood mononuclear cells and T cells. It was found that treatment with HDACi and c-FLIPi induced apoptosis and prevented proliferation of activated lymphocytes and T cells. Secondly, the mechanism by which HDACi and c-FLIPi induce apoptosis in these cells was investigated. Co-treatment of murine lymphocytes with the HDACi Entinostat alongside the caspase-8 inhibitor Z-IETD-FMK resulted in diminished incidence of apoptosis, suggesting that apoptosis induced by Entinostat is mediated by caspase-8, which may be the result of diminished c-FLIP activity. Further investigation is necessary to elucidate if apoptosis induced by c-FLIPi may be similarly attenuated. Similarly, further investigation is necessary to determine whether inhibition of death receptor signalling may prevent apoptosis induced by HDACi and c-FLIPi. Thirdly, it was found that the survival of murine skin allografts was extended following treatment with Entinostat, but no prolongation was observed following c-FLIPi treatment. Flow cytometry of skin, spleen and lymph tissue from graft recipients did not show a reduction in the T cell populations found within these tissues following Entinostat treatment, but rather an increase in the proportion of cytotoxic T cells in the lymph nodes of treated mice compared to controls. This suggests that Entinostat may prolong graft survival via a mechanism different from the induction of alloreactive T cell apoptosis observed in vitro, such as altering cytokine expression or increasing regulatory T cell populations or effectiveness.

These results suggest that the effects of c-FLIPi and HDACi are complex and, whilst initial results from in vitro models are promising, much work remains to be done to elucidate the roles and mechanisms of action of these drugs in the context of transplantation. The studies laid out in this thesis establish a foundation for further investigation into the potential of c-FLIP inhibition and HDACi utilisation in organ transplantation.

Date of Award	Dec 2023
Original language	English
Awarding Institution	Queen's University Belfast
Sponsors	Northern Ireland Kidney Research Fund & Northern Ireland Department for the Economy
Supervisor	James McDaid (Supervisor), Beckie Ingram (Supervisor) & Daniel Longley (Supervisor)