Endomucin marks quiescent long-term multi-lineage repopulating hematopoietic stem cells and is essential for their transendothelial migration.

Endomucin (EMCN) currently represents the only hematopoietic stem cell (HSC) marker expressed by both murine and human HSCs. Here, we report that EMCN+ long-term repopulating HSCs (LT-HSCs; CD150+CD48-LSK) have a higher long-term multi-lineage repopulating capacity compared to EMCN- LT-HSCs. Cell cycle analyses and transcriptional profiling demonstrated that EMCN+ LT-HSCs were more quiescent compared to EMCN- LT-HSCs. Emcn-/- and Emcn+/+ mice displayed comparable steady-state hematopoiesis, as well as frequencies, transcriptional programs, and long-term multi-lineage repopulating capacity of their LT-HSCs. Complementary functional analyses further revealed increased cell cycle entry upon treatment with 5-fluorouracil and reduced granulocyte colony-stimulating factor (GCSF) mobilization of Emcn-/- LT-HSCs, demonstrating that EMCN expression by LT-HSCs associates with quiescence in response to hematopoietic stress and is indispensable for effective LT-HSC mobilization. Transplantation of wild-type bone marrow cells into Emcn-/- or Emcn+/+ recipients demonstrated that EMCN is essential for endothelial cell-dependent maintenance/self-renewal of the LT-HSC pool and sustained blood cell production post-transplant.

Inhibition of Oxidized Nucleotide Sanitation By TH1579 and Conventional Chemotherapy Cooperatively Enhance Oxidative DNA Damage and Survival in AML.

Currently, the majority of patients with acute myeloid leukemia (AML) still die of their disease due to primary resistance or relapse toward conventional reactive oxygen species (ROS)- and DNA damage-inducing chemotherapy regimens. Herein, we explored the therapeutic potential to enhance chemotherapy response in AML, by targeting the ROS scavenger enzyme MutT homolog 1 (MTH1, NUDT1), which protects cellular integrity through prevention of fatal chemotherapy-induced oxidative DNA damage. We demonstrate that MTH1 is a potential druggable target expressed by the majority of patients with AML and the inv(16)/KITD816Y AML mouse model mimicking the genetics of patients with AML exhibiting poor response to standard chemotherapy (i.e., anthracycline & cytarabine). Strikingly, combinatorial treatment of inv(16)/KITD816Y AML cells with the MTH1 inhibitor TH1579 and ROS- and DNA damage-inducing standard chemotherapy induced growth arrest and incorporated oxidized nucleotides into DNA leading to significantly increased DNA damage. Consistently, TH1579 and chemotherapy synergistically inhibited growth of clonogenic inv(16)/KITD816Y AML cells without substantially inhibiting normal clonogenic bone marrow cells. In addition, combinatorial treatment of inv(16)/KITD816Y AML mice with TH1579 and chemotherapy significantly reduced AML burden and prolonged survival compared with untreated or single treated mice. In conclusion, our study provides a rationale for future clinical studies combining standard AML chemotherapy with TH1579 to boost standard chemotherapy response in patients with AML. Moreover, other cancer entities treated with ROS- and DNA damage-inducing chemo- or radiotherapies might benefit therapeutically from complementary treatment with TH1579.

Targeted inhibition of cooperative mutation- and therapy-induced AKT activation in AML effectively enhances response to chemotherapy.

Most AML patients exhibit mutational activation of the PI3K/AKT signaling pathway, which promotes downstream effects including growth, survival, DNA repair, and resistance to chemotherapy. Herein we demonstrate that the inv(16)/KITD816Y AML mouse model exhibits constitutive activation of PI3K/AKT signaling, which was enhanced by chemotherapy-induced DNA damage through DNA-PK-dependent AKT phosphorylation. Strikingly, inhibitors of either PI3K or DNA-PK markedly reduced chemotherapy-induced AKT phosphorylation and signaling leading to increased DNA damage and apoptosis of inv(16)/KITD816Y AML cells in response to chemotherapy. Consistently, combinations of chemotherapy and PI3K or DNA-PK inhibitors synergistically inhibited growth and survival of clonogenic AML cells without substantially inhibiting normal clonogenic bone marrow cells. Moreover, treatment of inv(16)/KITD816Y AML mice with combinations of chemotherapy and PI3K or DNA-PK inhibitors significantly prolonged survival compared to untreated/single-treated mice. Mechanistically, our findings implicate that constitutive activation of PI3K/AKT signaling driven by mutant KIT, and potentially other mutational activators such as FLT3 and RAS, cooperates with chemotherapy-induced DNA-PK-dependent activation of AKT to promote survival, DNA repair, and chemotherapy resistance in AML. Hence, our study provides a rationale to select AML patients exhibiting constitutive PI3K/AKT activation for simultaneous treatment with chemotherapy and inhibitors of DNA-PK and PI3K to improve chemotherapy response and clinical outcome.