FLT3-ITD drives poor prognosis in NPM1 mutated AML via BCAT1-dependent epigenetic regulation Free

NPM1 mutations (NPM1^mut) are detected in approximately 30% of adult AML cases. Patients with coexisting NPM1 mutations and FLT3-ITD mutations (NPM1^mutFLT3^ITD) exhibit significantly poorer clinical outcomes, while the FLT3-ITD allelic ratio (AR) shows limited utility for risk stratification in this double-mutant subgroup. In our previous studies investigating novel prognostic biomarkers for NPM1^mutFLT3^ITD patients, integrated analysis of RNA-Seq data and public databases demonstrated that branched-chain amino acid transferase 1 (BCAT1) was significantly upregulated in NPM1^mutFLT3^ITD AML cases. BCAT1 regulates branched-chain amino acids and inhibits the activity of DNA demethylase TET2 by reducing α-ketoglutarate (α-KG) levels. Herein, we further investigated the mechanistic role of BCAT1 in NPM1^mutFLT3^ITD AML.

Analysis of the BloodSpot database demonstrated predominant BCAT1 expression in hematopoietic stem/progenitor cells (HSPCs) of healthy individuals, with marked downregulation during differentiation. scRNA-seq of de novo AML cases (GSE198681) showed BCAT1 expression in leukemic stem/progenitor cells. Comparative analysis of NPM1^mut and NPM1^mutFLT3^ITD samples (GSE15434) revealed exhibited BCAT1 upregulation and enrichment in amino acid metabolism and DNA methylation pathways. FLT3-ITD knock-in OCI-AML3 cells (with NPM1^mut) demonstrated significant upregulation of BCAT1 expression, while AC220 (quizartinib) treatment substantially reduced BCAT1 expression. Probe-based RT-qPCR demonstrated significantly higher BCAT1 expression in NPM1^mutFLT3^ITD patients compared to NPM1^mut cases (56% vs 20%, ABL-normalized, p<0.0001), with median expression in healthy controls at only 6%. BCAT1 overexpression in OCI-AML3 cells accelerated proliferation, reduced apoptosis, promoted G2/M progression, and enhanced clonogenic capacity. RNA-seq analysis revealed BCAT1-mediated upregulation of proliferation, cell cycle, DNA repair, and stemness pathways, while downregulating immune chemotaxis and MHC class I/II expression. Integrated RNA-Seq and reduced representation bisulfite sequencing (RRBS) analyses of FLT3-ITD knock-in and BCAT1-overexpressing cell lines identified two tumor suppressor genes (AATK and NTNG2) that were epigenetically silenced through promoter hypermethylation. AATK (apoptosis-associated tyrosine kinase), involved in growth arrest and apoptosis, showed decreased expression across multiple cancers. RNA-seq of patients in our center confirmed significant AATK downregulation in NPM1^mutFLT3^ITD patients versus NPM1^mut cases (NPM1^mutFLT3^ITD patients vs NPM1^mut patients: p=0.002). BCAT1 knockout (BCAT1-KO) in MOLM13 cells (GSE100778) partially restored AATK expression (BCAT1-KO vs BCAT1-wt: 85.3 FPKM vs 83.4 FPKM, p=0.1), while BCAT1 overexpression (BCAT1-OE) increased AATK promoter methylation level(BCAT1-OE vs BCAT1-wt: Δβ > 0.1, p=0.013, GSE100782). Previous studies established that AATK phosphorylates TP53 to modulate its transcriptional activity. Analysis of published AATK-knockdown datasets in 293T cells confirmed that AATK depletion downregulates TP53 target genes, with enrichment in pathways related to cell proliferation, intrinsic apoptosis, and DNA damage repair, ultimately leading to accelerated tumor cell growth, enhanced survival under stress conditions, and accumulation of genomic instability, collectively contributing to oncogenic progression.

Our findings demonstrate that FLT3-ITD upregulates BCAT1 in NPM1-mutated AML, promoting proliferation and clonogenicity while epigenetically silencing AATK through hypermethylation. On one hand, AATK silencing (as a key apoptosis regulator) leads to uncontrolled cell proliferation and reduces apoptosis; on the other hand, diminished TP53 phosphorylation by AATK impairs its tumor-suppressive function. These combined effects render NPM1^mutFLT3^ITD cells apoptosis-resistant yet paradoxically vulnerable to apoptosis-inducing agents, providing a possible mechanistic rationale for combining hypomethylating agents with Bcl-2 inhibitors in this AML subtype. Furthermore, compromised TP53-mediated DNA damage response suggests potential susceptibility to DNA repair inhibitors (such as PARP inhibitors) in this patient subset, offering novel therapeutic opportunities.