Tyrosine kinase targeted (TKI) therapies have revolutionized leukemia treatment, but TKIs are not able to kill leukemia stem cells (LSCs), which are responsible for propagating and disease recurrence, and believed to be the source of treatment failure. Our long-term goals are to further address how LSC persistence is regulated, and to develop new LSC-eliminating treatment strategies to improve cure rates and survival. The short-term goals of this research are to determine whether a N6-methyladenosine (m6A)--long non-coding RNA (lncRNA) axis regulates LSC stemness and persistence during TKI selection process, and to explore the therapeutic potential of targeting the m6A-lncRNA axis for eradicating TKI resistant LSCs and also decipher the underlying molecular mechanisms. The m6A methylation is the most common epitranscriptomic modification on RNAs (i.e., lncRNAs), and crucially regulates lncRNA-initiated gene expression. lncRNA abnormalities frequently associate with cancer disease progression and drug resistance. The preliminary evidence linking an m6A-lncRNA axis to resistant LSCs is from our proof of principle studies demonstrating that i) a dynamic and reversible m6A methylome determined by fat mass and obesity-associated protein (FTO) helps leukemia cells avoid TKI killing leading to TKI resistance; ii) there are many lncRNAs (annotated) that are differentially expressed in resistant versus sensitive cells. About 50% of these lncRNAs bear m6A motifs and have the changed m6A amounts in resistant cells, collectively, suggesting a unique lncRNA signature that is specific to TKI resistance and is regulated by m6A methylation; iii) upregulation of these m6A-associated lncRNAs in patients who do not respond to TKIs is predicative of worse outcomes, and knockdown of them impairs resistant cell growth and renders resistant cells sensitive to TKIs; iv) compared to sensitive ones, TKI-resistant cells highly express LSC markers (CD117, CD44, CD25, CD133) whose upregulation is associated with m6A reduction. Our hypothesis is that the FTO-m6A-lncRNA cascade may be a critical pathway to control LSC persistence to TKIs and a new druggable target to eradicate persistent LSCs improving TKI cure rates. We will test our hypothesis through three aims: 1) Determine how the FTO-m6A axis regulates lncRNA aberrations in TKI resistance; 2) Determine whether and how a dynamic m6A methylome regulates LSC persistence to TKIs; 3) Determine whether and how pharmacological targeting of the FTO-m6A-lncRNA cascade kills persistent LSCs using preclinical leukemia models. The proposed studies are conceptually innovative, because it targets a new pathway (the FTO-m6A- lncRNA cascade) in understanding LSC persistence to TKIs and in developing new regimens to eliminate TKI resistant LSCs. The proposed research is significant, because the findings will a) identify new pathways (i.e., FTO-m6A-lncRNA cascade) that regulate LSC persistence, deepening the molecular understanding of LSC...