Mechanisms Of Targeted Mitochondrial Metabolic Intervention In Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is a highly heterogeneous hematologic malignancy.The development of molecular diagnostic technique,improved understanding of the biological function of AML and updated clinical treatment options in the last decade have greatly expanded the pathophysiologic typing of AML.Current epidemiological studies show that although targeted drugs and immunotherapies for AML are rapidly progressing and have improved patient prognosis and survival to some extent,the need for more precise and personalized treatment with less toxic side effects remains a challenge in the treatment of AML.Approximately 80% of ATP in human cells is produced by mitochondria,which involves two major pathways: the tricarboxylic acid cycle(TCA cycle)and the oxidative phosphorylation pathway(OXPHOS)powered by the electron transport chain(ETC).ETC pathway requires an adequate and stable supply of nicotinamide adenine dinucleotide(NADH)and flavin adenine dinucleotide(FADH2),which are produced through the TCA cycle.Correspondingly,an adequate supply of oxaloacetate and acetyl coenzyme A is required for the stable operation of the TCA cycle.Cells produce acetyl coenzyme A through three different catabolic pathways: the carbohydrate pathway,the lipid pathway and the protein pathway.Therefore,the link between OXPHOS and other substance metabolic pathways is essential for normal cellular energy production.Studies have shown that AML cells have a higher mitochondrial mass compared to normal hematopoietic cells and that AML cell proliferation is associated with a high metabolic phenotype,which includes increased basal and maximal mitochondrial respiration.Although mitochondrial mass is increased in AML,their mitochondrial respiratory chain activity is not significantly increased,which leads to increased susceptibility of AML cells to mitochondrial oxidative stress.Also,compared to normal hematopoietic cells,some AML cells exhibit upregulation of mitochondrial DNA(mt DNA)synthesis pathways,which are positively correlated with the OXPHOS.Previous studies on chidamide have focused more on its anti-leukemic effects through epigenetic histone modifications.In Chapter 3 of this thesis,we found that AML patients with high OXPHOS status were associated with poor prognosis and low survival,and had high intracellular expression of HDAC class I family proteins.Chidamide selectively inhibited HDAC class I family,especially HDAC1/3 protein expression.Chidamide suppressed AML cell proliferation and promoted apoptosis.Furtherly,mitochondrial function assays were also verified and the results showed that chidamide disturbed the balance of mitochondrial homeostasis and reduced mitochondrial metabolic function,as evidenced by the substantially lower oxygen consumption value(OCR)and reduced ATP production.In addition,chidamide inhibited the intracellular NF-k B pathway and reduced the expression of inflammatory factors.In the MLL-AF9-based AML mice model,chidamide also prolonged the survival of mice.AML patients with NPM1 mutations account for approximately 1/3 of newly diagnosed patients in adults.The unique clinical features and molecular pathology of NPM1 have led to its identification as a unique class of entities in the 2016 WHO myeloid hematologic tumor typing.NPM1 mutations are also one of the ideal targets for detection of microscopic residual disease(MRD),as the occurrence of NPM1 mutations in AML is specific and stable.New drugs with potential for future targeted therapies in NPM1 mutated AML are including nuclear export inhibitors(XPO1),MLL inhibitors(Menin),FLT3 inhibitors,and BCL-2 inhibitors(venetoclax).In Chapter 4 of this paper,we identified an abnormally high OXPHOS status in patients with NPM1 mutations by RNA-seq combined with Single Cell-seq,and the mutant cells were accompanied by increased leakage of mitochondrial DNA(mt DNA)into the cytoplasm.The leaked mt DNA bound to the classical ds DNA sensor c GAS in the cytoplasm,which in turn activated the downstream STING pathway,ultimately leading to elevation of the interferon-stimulated genes(ISGs).Since the structure of NPM1 protein is rich in high protein disorder region(IDR),we then conducted a series of classical experiments of liquid-liquid phase separation and found that wild-type NPM1 localized in the nucleus and underwent liquidliquid phase separation,which did not bind to c GAS.Mutant NPM1 did not undergo phase separation but could bind to c GAS,due to its aberrant localization in the cytoplasm caused by the wrong signal peptide at the C-terminus of the protein.NPM1 mutant cells were more likely to induce STING-dependent pyroptosis after the treatment of conventional AML chemotherapeutic drug cytarabine.This study provides a theoretical basis for the clinical precise treatment of NPM1 mutant patients.