Growth Factor Independence 1 (GFI1) is a zinc-finger transcriptional repressor and master regulator of growth, differentiation and survival in hematopoiesis. By repressing genes that favor cell cycle arrest and apoptosis, GFI1 confers a growth and survival advantage to cells that express it. We have discovered that NOTCH1 intracellular domain (N1ICD) binds and stabilizes GFI1. N1ICD shields a consensus element for SUMOylation embedded within the GFI1 linker, preventing SUMO-dependent polyubiquitination and degradation of GFI1. Thymoctyes require GFI1 to withstand Notch activation during normal T-lymphopoiesis, while mutations that favor unbridled Notch signaling cause T-cell acute lymphoblastic leukemia (T-ALL). GFI1 depletion triggers apoptosis in T-ALL cells. These data implicate N1ICD?GFI1 binding as a pro-survival adaptation to constitutive Notch activation and define GFI1 effectors and their recruitment mechanisms as novel, non- redundant therapeutic inputs for T-ALL. GFI1's dominant effector is Lysine Specific Demethylase 1 (LSD1). GFI1?LSD1 binding requires the GFI1 SNAG domain. Our proposed experiments focus on molecular determinants of LSD1 recruitment by GFI1 and test LSD1 inhibition as a strategy to treat T-ALL.
Aim #1 : Identify and define mechanisms governing integrity and function of the N1ICD?GFI1?LSD1 complex. The GFI1 SNAG domain is required for GFI1?LSD1 binding, but alone it has limited affinity. However, methylation on lysine (K) 8 in the -8KSKK11- motif of the SNAG domain profoundly favors SNAG?LSD1 binding. We will test the impact of K8 methylation on GFI1 function, identify methyltransferase(s) responsible and determine how LSD1 inhibition modulates binding relationships among components of the complex. This data informs mechanisms regulating complex integrity/function and may define an alternate inhibitory input for the GFI1?LSD1 axis.
Aim #2 : Determine efficacy of the LSD1 inhibitor, SP-2577, toward T-ALL in vitro and in vivo and identify candidate biomarkers of SP-2577 action. LSD1 inhibition is a rationally conceived strategy to treat T-ALL. SP-2577 is a potent, selective, and reversible inhibitor of LSD1 developed at Huntsman Cancer Institute that displays anti-proliferative, pro-apoptotic activity toward T-ALL cells. We will determine its efficacy toward primary patient specimens and identify/validate biomarkers indicative of SP-2577 activity.
Aim #3 : Determine the impact of GFI1?LSD1 binding on GFI1 pro-survival effects and SP-2577 efficacy in T-ALL. GFI1 depletion and LSD1 inhibition phenocopy one another in growth and differentiation assays. Using GFI1 and LSD1 derivatives that uncouple GFI1?LSD1 binding from LSD1 catalytic activity, we will determine if GFI1 pro-survival effects and SP-2577 efficacy depend upon GFI1 bound to catalytically active LSD1. These data inform mechanisms of cell survival in T-ALL and instruct us about the GFI?LSD1 axis as a target of LSD1 inhibitors. Our studies provide critical insights into an important functional axis in T-ALL while simultaneously advancing an innovative therapeutic strategy for this life-threatening cancer.
T-cell acute lymphoblastic leukemia (T-ALL) is a life-threatening cancer striking children and adults, yet despite aggressive treatment, over 500 people die from T-ALL each year in the United States and many more around the world. Knowing how T-ALL cells stay alive will allow us to find more effective, less toxic treatments for the disease. We have discovered one strategy that T-ALL cells use to stay alive, and through our experiments we will reveal how the strategy works and test a novel new drug that blocks it to kill T-ALL cells.
