Deciphering the role of MLL-partial tandem duplication in AML through integrated genomic and proteomic approaches

PROJECT SUMMARY/ABSTRACT Partial tandem duplication (PTD) of the N-terminal (N-term) region of the mixed lineage leukemia (MLL) gene (MLLPTD) is associated with a poor prognosis in acute myeloid leukemia (AML) and was recently identified as one of the strongest predictors of adverse outcome in myelodysplastic syndromes (MDS). The mechanism by which MLLPTD promotes leukemogenesis remains poorly understood, particularly in patients harboring complex alleles with three or more tandem repeats of the N-term domain, which correlate with more aggressive disease and higher relapse rates. There is a critical need to understand how MLLPTD repeat expansion alters protein function, chromatin binding, and transcriptional regulation, and to identify molecular vulnerabilities that can be therapeutically targeted. The long-term goal of this research is to elucidate how oncogenic transcription factors such as MLLPTD drive hematologic malignancies. The overall objective of this project is to leverage newly developed tools—including specific antibodies and targeted mass spectrometry assays—to define how allelic complexity modifies MLLPTD’s protein interactions and gene regulatory functions, and to identify downstream dependencies that sustain disease. The central hypothesis is that N-term repeat expansion promotes broader and mislocalized genomic occupancy, aberrant recruitment of epigenetic cofactors, and activation of pro-leukemic transcriptional programs, leading to progressively more severe disease. The rationale is that identifying the protein interactors and genomic targets associated with different MLLPTD variants will yield key mechanistic insights and reveal actionable vulnerabilities. Three specific aims are proposed: Aim 1. Characterize the MLLPTD protein and its interactome in AML patients. Targeted mass spectrometry will be used to quantify protein abundance and Nterm repeat complexity in AML cells, and immunopurification followed by mass spectrometry will identify interacting partners, including cofactors enriched in high-repeat variants. Aim 2. Determine how expansion of the Nterm domain of MLL alters its gene regulatory function. CUT&Tag profiling and functional perturbations in AML cells will assess how repeat expansion impacts genomic binding, histone modifications, and transcriptional output. Aim 3. Functionally validate novel therapeutic target pathways. Key MLLPTD-associated factors will be perturbed using genetic and pharmacologic approaches in patient-derived xenograft (PDX) models to evaluate their role in leukemia maintenance and therapeutic response. PDX models are required to assess leukemia-initiating capacity and therapeutic response in vivo, which cannot be recapitulated in vitro. This project is innovative in its focus on allelic complexity as a driver of leukemogenesis and in its integration of proteomic, transcriptomic and epigenomic technologies to dissect MLLPTD function. It is significant because it will establish a mechanistic framework for understanding MLLPTD-driven disease and identify new strategies for targeted therapy in poor-prognosis AML and MDS.