MLL5-Driven Leukemic Transformation Is Linked to Chromatin Remodeling and Can be Targeted with ATRA in Combination with PARP1 Inhibition
Authors: Simoes Luise Araujo De Albuquerque , Weinhäuser Isabel , Pereira-Martins Diego A , Almeida Luciana Yamamoto , Van Den Boom Vincent , Mojallali Fatemeh , Sternadt Dominique , Chatzikyriakou Prodromos , Zheng Xuesen , Ortiz Rojas César Alexander , Mufti Ghulam Jeelani , Pinheiro Lucio Henrique , Quek Lynn , Huls Gerwin A. , Rego Eduardo M. , Schuringa Jan Jacob 
Published: December 2024

The mixed lineage leukemia (MLL) genes are part of the trithorax group of proteins, which play a crucial role in the regulation of genes that control self-renewal and differentiation. In acute myeloid leukemia (AML) patients, high expression of MLL5 (KMT2E) is linked to a higher disease burden. Yet, the precise mechanisms by which MLL5 influences AML progression and response to therapy remain unclear. To study the impact of MLL5 on leukemogenesis in vivo we developed three independent cell line-derived xenograft (CLDX) mouse AML models. We consistently observed superior tumor burden upon overexpression (OE) of MLL5. To elucidate underlying mechanisms that mediate MLL5-driven leukemic transformation we performed single-cell RNA sequencing in a panel of primary AML patient samples. While MLL5 was expressed across all myeloid cell compartments, its expression was highest in the leukemic stem cell (LSC) population. We therefore focused on the LSC populations and compared the gene expression profiles of MLL5high versus MLL5low LSCs. We identified 69 genes that were upregulated in MLL5high LSCs, associated with histone acetyltransferase activity, chromatin remodeling, and DNA/RNA binding. ChIP-seq analyses were performed to explore how MLL5 influences the epigenetic landscape and gene expression. In several MLL5-OE AML models, we consistently observed an increased deposition of H3K27ac marks at gene promoter regions compared to empty vector (EV) controls. Gene set enrichment analyses revealed an association of these H3K27ac-enriched loci with terms “MYC_TARGETS”, CELL_CYCLE“, ”DNA_DAMAGE_REPAIR“, and ”OXIDATIVE_PHOSPHORYLATION (OXPHOS)“. Although global H3K4me3 distribution did not show significant changes between the two groups, specific genes with increased H3K4me3 were identified MLL5-OE cells, including PARP1, MYC, and LDHA, linked to ”CELL_CYCLE“ and ”OXPHOS“. Molecular profiling of a cohort of AML patients (n=823) indicated that MLL5high AML patients are enriched with primed LSPCs, and molecular programs linked with ”MYC_TARGETS“, ”PARP1_SIGNALING“ and ”OXPHOS“. These findings collectively suggest that MLL5-OE drives a proliferative phenotype in AML, with increased reliance on DNA damage repair and OXPHOS pathways. Lentiviral MLL5-OE resulted in enhanced cell proliferation and imposed drug resistance against standard-of-care cytotoxic therapies in AML (e.g., Cytarabine and Venetoclax, VEN), alongside an increase in functional respiration capacity. Conversely, MLL5-OE was associated with increased sensitivity to epigenetic/differentiation agents like All-trans retinoic acid (ATRA), Decitabine, and Azacytidine (AZA), as well as to PARP1 inhibitors AG14361 and Olaparib, typically used for solid tumors. Further validation using two cohorts of ex vivo treated primary AML samples (our cohort (n=25) and 2nd cohort (n=30, Lee et al., Nat Comm 2015), showed that MLL5high samples were more responsive to differentiation-inducing agents, including ATRA and AG14361, with a notable increase in the DNA damage marker γH2AX upon treatment. ATRA treatment of primary CD34+ AML blasts was associated with a significant reduction in mitochondrial membrane potential, suggesting effects on OXPHOS metabolism. Additionally, the combination of ATRA with PARP1 inhibitors AG14361/Olaparib synergistically induced apoptosis in MLL5-OE AML cells. In vivo, mice transplanted with MLL5-OE cells showed increased sensitivity to ATRA treatment, and TUNEL analysis of the retrieved tumors revealed increased apoptosis in MLL5-OE cells treated with ATRA. These effects were abrogated upon knockdown of MLL5. In summary, our study demonstrates that MLL5 plays a critical role in chromatin remodeling in AML, leading to a more open chromatin state and the upregulation of genes linked to cell cycle progression and OXPHOS-metabolism, thereby reinforcing its function as an oncogene in AML. Paradoxically, while MLL5-OE enhances resistance to cytotoxic therapies, it also creates a vulnerability to differentiation agents and PARP1 inhibitors. Our findings suggest that a combination therapy using ATRA with PARP1 inhibitors represents a promising therapeutic option for MLL5high AML patients that should be further exploited.