CLINICAL SIGNIFICANCE OF SOMATIC MOSAIC STATES IN THE CONTEXT OF SHORT TELOMERES - (PLEASE KEEP IN WORD, DO NOT PDF) Telomere biology disorders (TBD) are multisystem disorders caused by premature shortening of telomere lengths. Hematopoietic stem and progenitor cells (HSPC) in TBD show impaired proliferative capacity manifesting as bone marrow failure (BMF). We and others have shown that HSPCs from TBD patients may develop somatic mosaic events that lead to context-specific clonal hematopoiesis (CH), overcoming replicative constraints. Due to TBD-specific unique stressors, these patients have a higher prevalence of CH (40% vs. 6% in age-matched non-TBD individuals) and a characteristic context-specific mutational spectrum mainly in DNA damage response and repair (DDRR) pathways and the p.S34 position in the spliceosome gene U2AF1, which results in multiple missplicing events. U2AF1MT-S34 is associated with rapid clonal expansion and evolution to myeloid neoplasm in non-TBD population, but TBD-U2AF1MT-S34 patients show a perplexing stability over time. This suggests that clonal dynamics and clinical outcomes of CH in TBD differ from age-related CH, with the underlying mechanisms behind these differences still unknown. We hypothesize that U2AF1MT-S34 somatic events result in splicing alterations in key pathways that rescue hematopoiesis in the context of TBD and may lead to adaptive and/or maladaptive outcomes, including progression to myeloid neoplasm. To test this, we will create and characterize a new K562 hematopoietic stem cell line to study our preliminary observations in TBD patients with U2AF1MT-S34 CH. In Aim 1, we will use a novel CRISPR/Cas9 method developed in our laboratory to create a K562 TERTMT cell line. We have used this method successfully in HEK-293t cells resulting in a phenotype that resembles TBD (low telomerase activity, short telomeres, replicative impairment). Using the same approach, we will introduce the U2AF1MT-S34 variant in this K562 TERTMT cell line and characterize the effect of these co-occurring mutations on telomere length, replicative capacity and morphology. In Aim 2, we will characterize the transcriptomic missplicing changes exerted by U2AF1MT-S34 in relevant pathways (DDRR, splicing, and telomere maintenance) in the context of short telomeres using long-read RNA sequencing (Oxford Nanopore). This project offers both conceptual and methodological innovation by creating a new model of TBD-CH and using it to characterize the downstream effect of the most frequent somatic event observed in these patients.
