Molecular basis of autophagy dysfunction in ß-thalassemia, drug regulatable pathways for ameliorating disease severity

ß-thalassemia is a red blood cell (RBC) disease, and one of the most prevalent hereditary disorders globally. It is a major source of morbidity and mortality. The disease is characterized by reduced or absent ß-globin, a critical component of the haemoglobin tetramer in RBCs that transports oxygen around the body. This causes an excess of the other tetramer component, a-globin, which forms insoluble aggregates. These trigger a series of downstream events that cause impaired maturation and death of the developing erythroid cells. This condition is termed ‘ineffective erythropoiesis’ (IE), the hallmark and major cause of ß-thalassemia disease pathophysiology. The estimated annual birth rate of infants with ß-thalassemia is 70,000-100,000 with a global prevalence of 300,000 for severe ß-thalassemia. By far the majority of patients are in low- and middle-income countries (LMIC). However, there are few treatment options. The mainstay is life-long or intermittent blood transfusion, but this has associated complications of alloimmunisation and iron-mediated potentially fatal organ damage, requiring life-long iron-chelation therapy. The main focus of therapeutic development has been gene therapy with two recently licensed (Casgevy and Zynteglo). However, although important, the complexity, extremely high costs and necessity for specialist facilities, mean they are unlikely to scale to the massive global unmet need for ß-thalassemia therapeutics, particularly in LMIC, with efficacy and safety issues also still concerns. One drug, Reblozyl® was licensed in 2020, but is only effective in some patients. Thus, therapeutic targets and cost-effective, easily assessable drugs are desperately required to deliver therapies to the greatest numbers of people. The ULK1-dependent autophagy pathway is essential for removing toxic a-globin aggregates in erythroid cells. However, whether this pathway functions normally in ß-thalassemia erythroid cells is unclear, although data indicating pathologically increased mTORC1 signalling in these cells correlates with inhibited autophagy. This provides drug-regulatable routes for activating or amplifying autophagy to remove the a-globin aggregates and ameliorate ß-thalassemia disease severity. We recently created model erythroid cell lines for ß-thalassemia that accurately recapitulate the disease phenotype of patient erythroid cells, providing a sustainable and consistent supply of cells for studying the disease. We plan to use the lines to perform in depth and definitive analysis of autophagy status in ß-thalassemia erythroid cells, along with analysis of approaches to increase autophagic flux via modulation of regulatory pathways and identification of targeting drugs to reduce disease severity. To achieve we will: 1) Definitively determine autophagy status in ß-thalassemia erythroid cells, using established markers, reporter cell-lines and quantitative electron microscopy for definitive analysis of autophagic flux. 2) Explore a range of directed approaches targeting the mTORC1 activator OTUD5, mTORC1 itself, the AMPK pathway, and the key autophagy initiator ULK1, to determine if result in increased autophagic flux, reduced a-globin aggregates and thus reduced disease severity. 3) Take an unbiased approach, screening bespoke library of drugs that target proteins and pathways that regulate autophagy, and are licensed or in trial for other diseases, to identify those that ameliorate IE to take forward for repurposing for ß-thalassemia, with pre-clinical follow up of identified drugs. Overall, the project will increase understanding of molecular basis of b-thalassemia disease phenotype, reveal potential novel therapeutic targets, and determine if novel drugs and drugs that act to induce autophagy for other diseases have the potential to be repurposed for b-thalassemia.