The overarching theme of our research is to decipher the molecular events responsible for bone marrow failure (BMF) in patients with impaired telomere maintenance, with the goal of improving treatment outcomes in these patients. The focus of this proposal is to use human pluripotent stem cells (hPSCs) as a novel platform to understand the molecular regulation of definitive hematopoietic impairment in cells with disease-associated mutations in telomerase, and to find novel alternatives to increase hematopoietic output in cells with dysfunctional telomeres. Dyskeratosis congenita (DC) is an inherited BMF syndrome where patients have very short telomeres for their age, typically below the first percentile length when compared to the rest of the population. All mutations discovered in DC are in genes that affect telomere homeostasis, including mutations in the telomerase components TERT, TERC, and dyskerin (DKC1). Difficulties in isolating relevant cell populations from DC patients have precluded the development of therapies against this disease. We recently overcame this limitation and combined genome engineering in hPSCs with in vitro human hematopoietic differentiation methods to create a novel and robust platform to study the mechanisms of hematopoietic failure caused by telomere dysfunction. In this submission, three specific aims are proposed that utilize this system to understand the molecular consequences of telomerase impairment during hematopoietic development, and to decipher novel targets to rescue hematopoietic output in cells harboring eroded telomeres.
In aim 1, we will decipher the role of DNA damage responses (DDR) in the progression of definitive hematopoietic failure from cells with low levels of TERC. In particular, we will focus on the role of the p53 pathway in the hematopoietic impairment of cells with short telomeres, decipher the roles of miR-34a and miR-145 during abnormal hematopoiesis and decipher the genetic consequences of silencing DDR to restore hematopoietic output in cells harboring DC- associated mutations.
In aim two we will decipher if the modulation of RNA decay in cells harboring mutations in DKC1 is a viable strategy to restore hematopoietic output in these cells. We will modulate TERC posttranscriptional processing and degradation by inhibition of specific RNA decay pathways. Finally, in aim 3, we will investigate if novel, non-canonical roles of TERC outside telomere maintenance can influence hematopoietic output and potentiate disease phenotypes in patients harboring mutations that reduce TERC levels, since these are commonly afflicted with severe disease phenotypes. Collectively, the studies proposed will allow us to decipher novel targets for directed therapies in patients suffering with telomere-syndromes. Our unique cellular and molecular tools, combined with our expertise in telomerase, stem cell biology and hematopoiesis puts us in an ideal position to make a significant impact in this field.
Telomerase dysfunction has been implicated in the pathogenesis of a growing number of human diseases, including bone marrow failure. In this proposal, we will determine the molecular regulation of hematopoietic impairment caused by mutations in telomerase, and elucidate novel alternatives to improve treatment outcomes of patients suffering with this devastating condition. Using a combination of different genetic and biochemical tools, the experiments proposed will greatly increase our knowledge on stem cell function and regulation in bone marrow failure syndromes.
| Brestoff, Jonathan R; Vessoni, Alexandre T; Brenner, Kirsten A et al. (2018) Acute graft-versus-host disease following lung transplantation in a patient with a novel TERT mutation. Thorax 73:489-492 |
