Project Summary Chronic myeloid leukemia (CML) is an aggressive hematological malignancy caused by the oncoprotein BCRABL1. Although tyrosine kinase inhibitors (TKIs) can help CML patients achieve molecular remission, they cannot be fully cured because the leukemic stem cells (LSCs) are still present in the body. This application aims to study a novel actionable mechanism that controls self-renewal and survival in LSCs with the intent to support the early-stage development of LSC-specific frontline therapy that increases treatment-free remission upon TKI discontinuation. Our team discovered that the transcription factor KLF4 represses the gene encoding the dual- specificity tyrosine-phosphorylation regulated kinase DYRK2, allowing LSCs to self-renew and progress the disease. Thus, deleting the Klf4 gene in a CML mouse model was associated with a lower frequency of LSCs, leading to leukemia regression and DYRK2 protein upregulation. In addition, inhibiting the SIAH2 ubiquitin ligase also leads to upregulating the DYRK2 protein, which is associated with cytotoxicity in CML cells. Based on these findings, we hypothesize that DYRK2 is a critical checkpoint that inhibits the survival and self-renewal in LSCs that can be activated through genetic and pharmacological stabilization of the DYRK2 protein. In this application, we propose to investigate the regulation of DYRK2 in CML LSCs through genetic studies to validate this molecular target and identify DYRK2 stabilizers for drug development. In Specific Aim 1, we propose to study the mechanism of DYRK2 activation in LSC self-renewal through the phosphorylation of downstream targets. In Specific Aim 2, we will explore the genetic stabilization of the DYRK2 protein by deleting the gene encoding the ubiquitin ligase SIAH2 and performing a genome-wide CRISPR/Cas9 screen to identify pathways regulating DYRK2 expression. In Specific Aim 3, we propose investigating the anti-leukemic properties of pharmacological stabilization of DYRK2 protein with small molecules identified in a biased approach and cell-based screens in pre-clinical mouse models as single agents or combined with imatinib. These comprehensive studies will elucidate how DYRK2 regulates LSCs, how DYRK2 expression is regulated, and what small molecules stabilize DYRK2 protein for developing LSC-directed therapy in leukemia.