TGM2 as a novel therapeutic target in venetoclax-resistant AML: Disrupting histone serotonylation-driven mitophagy with nanoparticle-delivered inhibitors Free

TGM2 as a Novel Therapeutic Target in Venetoclax-Resistant AML: Disrupting Histone Serotonylation-Driven Mitophagy with Nanoparticle-Delivered Inhibitors Lu Lu^1*, Chunlei Dai^1*, Lezong Chen^1*, Xiao Zhang^1*, Yinglin Su¹, Zhenzhen Qiu¹, Jingxia Xu, Xumiao Zhang¹, Chenfei Liu¹, Langqi Wang¹, Yongshuai Jiang¹, Tao Chen¹, Yang Liang^1#, Yuanbin Song^1#

¹Department of Hematologic Oncology, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou, China.

*Co-first authors #Co-corresponding authors

Corresponding author：Yuanbin Song, Yang Liang.

Venetoclax (VEN) combined with hypomethylating agents is approved for frontline therapy in older/unfit patients with acute myeloid leukemia (AML). However, up-front resistance as well as relapse following initial response demonstrates the need for a deeper understanding of resistance mechanisms. Previous studies have revealed the critical role of transglutaminase 2 (TGM2) as a potential therapeutic target in cancers, but the oncogenic roles and underlying mechanisms of TGM2 in AML are not fully understood. In this study, we examined the role and potential mechanism of TGM2 in AML.

RNA-seq analysis revealed that TGM2 is overexpressed in AML patients, particularly in those with VEN resistance, and correlates with poor overall survival. Both CRISPR/Cas9-mediated knockout of TGM2 and pharmacological inhibition of TGM2 can inhibit leukemia stem cells (LSCs) derived from AML patients, including those resistant to VEN. Pharmacological inhibition of TGM2 demonstrated significant anti-leukemic activity in both MLL-AF9 and patient-derived xenograft (PDX) models.

Mechanistically, integrated analysis of RNA-seq and single-cell RNA sequencing (scRNA-seq) revealed that TGM2 inhibition perturbs mitochondrial homeostasis. Transmission electron microscopy (TEM) revealed that following TGM2 inhibition, mitochondria exhibited disrupted and swollen cristae, accompanied by a decrease in mitochondrial membrane potential and an increase in superoxide levels. Mitophagy, a key mechanism for intracellular homeostasis, critically supports LSC survival. By clearing damaged mitochondria and reducing ROS accumulation, LSCs maintain mitochondrial function and homeostasis, thereby enhancing their survival and therapy resistance. We demonstrated that targeting TGM2 can reduce mitophagic flux and block the autophagic flow. It was found that TGM2-mediated H3Q5ser histone modification regulates key gene expression programs by stabilizing H3K4me3 and enhancing its recognition by downstream effectors. CUT&Tag and RNA sequencing analyses revealed that downregulated genes were enriched in the Mitophagy pathway following treatment with TGM2 inhibitors. Western blot (WB), ChIP and qPCR assays demonstrated significantly reduced expression of key mitophagy genes (PINK1, Parkin, NDP56, and OPTN) following TGM2 inhibition.

Finally, to enhance bone marrow targeting of TGM2 inhibitors for effective elimination of LSCs, we developed a leucine polyester amide (Leu-PEA) nanoparticle drug delivery system (Leu-PEA@TGM2i). Leu-PEA@TGM2i significantly suppressed AML progression in both MLL-AF9 and PDX models without inducing appreciable myelosuppression or tissue damage.

In summary, TGM2 represents a potential prognostic biomarker in AML, and targeting its transglutaminase activity effectively inhibits disease progression. Specifically, TGM2 mediates H3Q5ser histone modification to promote expression of key mitophagy genes, thereby maintaining LSC function and inducing drug resistance. Inhibition of TGM2 disrupts mitochondrial homeostasis, providing a basis for novel therapeutic strategies.