Using rapid protein degradation to determine the effect of RUNX1 loss-of- function on DNA damage accumulation and repair.

Author

Jackriel Pina Morales, Lehman College City University of New YorkFollow

Date of Award

Spring 5-30-2024

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

First Advisor

Kristy Stengel

Second Advisor

Kristina Ames

Third Advisor

Stephen Redenti

Abstract

Germline mutations in RUNX1 are associated with familial platelet disorder with a predisposition to myeloid malignancy (RUNX1-FPDMM), in which patients present with low platelet counts,excessive bleeding and bruising, and an increased risk of Acute Myeloid Leukemia (AML)/Myelodysplastic Syndrome (MDS) development throughout their lifetime. To understand how these loss-of-function mutations in RUNX1 drive predispose to malignancy, it is important to develop a detailed understanding of the molecular basis of RUNX1 function. While RUNX1 is a transcription factor, preliminary data from our group and work from others suggests that RUNX1 also interacts with proteins that are critical for DNA damage repair. In addition, it has been observed that RUNX1 mutant patient cells have elevated levels of DNA damage, suggesting that a direct role for RUNX1 in DNA repair and maintenance of genome integrity could directly contribute to its tumor suppressive function. Thus, we hypothesize that RUNX1 is involved in DNA repair through either indirectly or directly through association with DNA repair proteins. However, it is very difficult to discern whether the loss of RUNX1 contributes to the accumulation of DNA damage directly due to a direct role in DNA repair, or whether it does so indirectly through the ability to regulate the expression of target genes that are critical for the DNA damage response. To distinguish between these two scenarios, we have used CRISPR-based genome editing to engineer AML cells to produce a degron-tagged RUNX1 protein. This allows us to treat cells with a small molecule proteolysis targeting chimera (PROTAC) called dTAG to rapidly degrade the endogenous RUNX1 protein from human leukemia cells. Rapid elimination of the RUNX1 protein (within 1hour) allows us to assay the effect of RUNX1 protein loss on DNA repair prior to the accumulation of large-scale transcriptional changes that occur at later timepoints following RUNX1 removal. Thus, we are poised to determine the mechanism(s) by which RUNX1 loss-of-function mutations contribute to the elevated DNA damage levels observed in patients and to determine to what extent DNA damage repair pathways may be directly impacted in these individuals. These findings will have clear implications for our understanding of the etiology of RUNX1 mutant myeloid malignancy and will have therapeutic implications given that standard of care often involves DNA damaging agents.

Recommended Citation

Pina Morales, Jackriel, "Using rapid protein degradation to determine the effect of RUNX1 loss-of- function on DNA damage accumulation and repair." (2024). CUNY Academic Works.
https://academicworks.cuny.edu/le_etds/37