Single-Cell Gene Expression Analyses Reveal Distinct Self-Renewing and Proliferating Subsets in the Leukemia Stem Cell Compartment in Acute Myeloid Leukemia

Abstract

Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with selfrenewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steadystate conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target selfrenewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/NRAS G12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes "LSCspecific" genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69 High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36 High LSCs were unable to transplant leukemia but were highly proliferative. These data dem-onstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target selfrenewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML.

EGR1 dysregulation defines an inflammatory and leukemic program in cell trajectory of human-aged hematopoietic stem cells (HSC)

“…Among alterations of the aged HSC phenotype, a myeloid-biased differentiation has been described [ 51 ]. In a recently described animal model of AML, leukemic stem cells characterized with a high expression of CD69 were found to be poorly proliferative and more capable self-renewal and [ 52 ], the same properties that we observed in the old human HSC compartment. TMEM107, a transmembrane molecule, is required for normal Sonic Hedgehog (shh) signaling in the development of the neural tube in combination with Gli2 and Gli3 to pattern ventral and intermediate neuronal cell types [ 53 ].…”

Escape From Treatment; the Different Faces of Leukemic Stem Cells and Therapy Resistance in Acute Myeloid Leukemia

“…LSCs can induce lipolysis in adipocytes to induce FAO in AML (stem) cells by abundant fatty acids, thereby evading chemotherapy-driven elimination of the AML cells ( Table 1 ). Only part of the LSC population in the BM express the fatty acid transporter CD36 ( 151 ), and these CD36+ LSCs showed to be highly proliferative and distinct from the pool of CD69 expressing LSCs that contain self-renewal potential ( 152 ). Further research should investigate if targeting the fatty acids uptake by CD36 would be a successful strategy to specifically eliminate AML LSCs.…”

Acute Myeloid Leukemia Stem Cells: The Challenges of Phenotypic Heterogeneity

“…Novel technical developments allowing high-throughput screening of low amounts of cells on both transcriptome [ 18 , 25 , 147 ], proteome [ 147 , 148 ], and surface antigen level [ 25 ] may provide further valuable insights into LSC surface antigens (e.g., identification of the fatty acid translocase CD36 and the type 2 C-lectin receptor CD69 via single-cell RNA sequencing [ 67 ]).…”