Somatic Truncation Mutations of the Histone Methyltransferase MLL4 Alter Its Subcellular Localization in Bladder Cancer: Mechanistic Insight and Targeted Therapeutic Approach

/ABSTRACT: KMT2D is subject to a high frequency of recurrent somatic mutations in many cancers, most notably bladder cancer (BLCa), and nearly 40% of KMT2D mutations lead to truncation or other major alteration of its product, the COMPASS monomethyltransferase MLL4. However, the tumorigenic mechanisms specific to MLL4 truncation have yet to be fully elucidated. The preliminary and published studies presented here reveal that truncated MLL4 consistently re-localizes from the nucleus to the cytosol, indicating the potential for non-canonical tumorigenic mechanisms arising from MLL4 truncation mutations, which could involve loss of catalytic-independent MLL4/COMPASS functions or a potential gain of function for truncated MLL4 in the cytoplasm. Importantly, cytoplasmic MLL4 is readily detectable in bladder tumors with MLL4 truncation. The goal of this application is therefore to accomplish three specific Aims: 1. Define the molecular mechanisms by which MLL4 truncation mutation and relocalization to the cytosol alter MLL4/COMPASS function to drive tumorigenesis, 2. Develop MLL4 mutation status and cytoplasmic localization as predictive biomarkers for patient stratification and targeted therapy in BLCa, and 3. Evaluate the impact of MLL4 mutation on response to pemetrexed treatment using in vivo animal models and patient-derived ex vivo models of MLL4/COMPASS- mutated BLCa. In Aim 1, we build upon preliminary findings of genomic instability due to transcriptional dysregulation in SET-deleted and UTX KO cells to propose ChIP-seq analysis of differential chromatin occupancy for nuclear MLL4/COMPASS factors such as UTX, with RNA-seq for associated differential gene expression, in isogenic MLL4 truncation vs WT cell lines. Based on preliminary findings of interaction between truncated MLL4 and the de novo nucleotide synthesis enzyme PAICS, we also propose to employ mass spectrometric analysis of MLL4 purified from these isogenic lines to identify and characterize novel, cytoplasmic protein interaction partners of truncated MLL4. These studies have the potential to deliver MLL4 truncation- specific targets for small molecule disruption, enabling development of targeted cancer therapies. In Aim 2, we propose to establish the association of cytoplasmic MLL4 IHC staining with MLL4 truncation mutation status in BLCa patient samples and perform a retrospective study exploring the potential use of MLL4 mutation status in BLCa for predicting responsiveness to the methotrexate-containing chemotherapy MVAC. These applied studies have the potential to enable patient stratification and informed treatment decision-making, advancing progress towards individualized BLCa treatment. In Aim 3, we build upon our recently reported finding of a targetable de novo nucleotide synthesis dependence in MLL4-mutated cancers, proposing studies to evaluate BLCa sensitivity to the de novo nucleotide synthesis inhibitor pemetrexed in vivo and to further evaluate MLL4 mutation-specific responsiveness to pemetrexed in autochthonous mouse and patient-derived organoid models of BLCa. These pre-clinical studies have the potential to support future clinical trials of targeted therapies for MLL4-mutated BLCa. Project Number: 1R01CA298333-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Ali Shilatifard (+1 co-PI) | Institution: NORTHWESTERN UNIVERSITY, CHICAGO, IL | Award Amount: $2,970,010 | Activity Code: R01 | Study Section: Advancing Therapeutics A Study Section [ATA] View on NIH RePORTER: https://reporter.nih.gov/project-details/11246158