Abstract B19: Lysine specific demethylase-1 inhibition as a therapeutic strategy that leverages the requirement for growth factor independence-1 in Notch-driven T-ALL

The zinc-finger (ZnF) transcriptional repressor Growth Factor Independence (GFI) 1 is a master regulator of lineage allocation in hematopoietic, aerodigestive tract and central nervous system development. Transcriptional repression by GFI1 requires a SNAG domain at its N-terminus and a concatemer of six ZnFs at its C-terminus. The SNAG domain recruits Lysine Specific Demethylase (LSD) 1, a dominant effector of transcriptional repression by GFI1, while ZnFs 3-5 bind a consensus response element in the promoters of GFI1 target genes. A linker region separates the SNAG and ZnF domains in GFI1, and likely provides sites for protein—protein interactions and post-translational modifications that modulate GFI1 functions in cell fate determination. In hematopoiesis, GFI1 regulates self-renewal and maintenance within the hematopoietic stem cell (HSC) compartment, supports early events in lymphopoiesis and is required for maturation of granulocytic precursors. Mutations in GFI1 cause severe congenital neutropenia (SCN) type II, a pre-leukemic condition, and recent evidence shows that GFI1 maintains the malignant phenotype in lymphoid leukemias and lymphomas. These findings intimate that regulators and effectors of GFI1 may be attractive candidates for therapeutic development in hematologic malignancy and maturational failure syndromes. How GFI1 is regulated to control cell fates during hematopoiesis is unknown. We show that GFI1 contains a consensus SUMOylation motif, centered on lysine (K) 239 within its linker region that is required for GFI1 conjugation by SUMO/Ubiquitin-like (UBL) proteins. K239 is found within the binding site for PIAS3, an E3 SUMO ligase and known GFI1 binding protein. PIAS3 antagonizes GFI1-mediated transcriptional repression. Arginine substitution at K239 (GFI1-K239R) preserves transcriptional repression by GFI1 yet resists the inhibitory effects of PIAS3. Moreover, the GFI1-K239R derivative displays a prolonged half-life and preferentially segregates with chromatin in fractionated cells. These findings suggest that GFI1-mediated transcriptional repression is limited by SUMO/UBL protein conjugation and that its roles in cell fate determination may be altered by factors governing these modifications. Among these factors is the Notch1 intracellular domain (N1-ICD), which we show interacts with the same region in GFI1 that binds PIAS3 and competes with PIAS3 for GFI1 binding. Furthermore, N1-ICD abolishes GFI1 conjugation by SUMO/UBL proteins, prolongs GFI1 half-life and reverses the inhibitory effect of PIAS3 toward GFI1-mediated transcriptional repression. In so doing, N1-ICD supports the function of GFI1 as a transcriptional repressor. We also show that Gfi1 depletion causes profound apoptosis in T-ALL cells driven by N1-ICD, suggesting GFI1 inhibitors and/or small molecule inhibitors of its effectors may prove efficacious in T-ALL treatment. To address this hypothesis, we have developed reversible small molecule inhibitors of LSD1. These inhibitors display sub-micromolar IC 50 toward T-ALL cell lines and primary patient isolates in the ex vivo setting, including those resistant to γ-secretase inhibitors. These findings suggest that the GFI1—LSD1 axis has significant influence over T-ALL viability and that LSD1 may be a novel therapeutic target in cancers driven by Notch activating mutations. Citation Format: Jason Singer, Daniel Andrade, Diana Bareyan, David McClellan, Helena Lucente, Matthew Velinder, Luke Maese, Mahesh Chandrasekharan, Emily Theisen, Fang Liu, Sunil Sharma, Michael Engel. Lysine specific demethylase-1 inhibition as a therapeutic strategy that leverages the requirement for growth factor independence-1 in Notch-driven T-ALL. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B19.