Single-cell gene expression analyses reveal distinct self-renewing and proliferating subsets in the leukemia stem cell compartment in murine acute myeloid leukemia

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 (LSCs), the cells with self-renewal capacity. Self-renewal and proliferation are mutually exclusive in normal hematopoietic stem cells (HSCs) in steady state conditions. If these functions are also mutually exclusive in LSCs, then antiproliferative therapies may fail to target self-renewal, allowing for relapse. We investigated whether proliferation and self-renewal are mutually exclusive in LSCs as they often are in HSCs. Using single-cell RNA sequencing, we identified distinct transcriptional profiles within LSCs of Mll-AF9/NRASG12V murine AML. We used single-cell qPCR and found 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 smaller subset of genes constitutes “LSC-specific” 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. Using in vivo mouse reconstitution assays, we found that only CD69High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36High LSCs were unable to transplant leukemia but were highly proliferative. These data demonstrate 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 self-renewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML. Overall design: Our study uses single-cell RNA sequencing to identify transcriptional heterogeneity within LSCs and define the self-renewing subset within this compartment. We performed single-cell RNA sequencing on the LSC-enriched compartment of our murine leukemia model. As we previously demonstrated that self-renewal is dependent on NRASG12V activity in this model, we also performed single-cell RNA sequencing on the LSC-enriched compartment after turning off NRASG12V transgene expression. To identify the functional contribution of each single-cell transcriptional profile, we performed in vivo assays of proliferation (using CellTrace labeling) and self-renewal (using in vivo leukemia reconstitution assays). We also performed single-cell qPCR on primary human LSCs and normal bone marrow HSPCs to determine whether these cells express components of this single-cell self-renewal signature. Finally, we analyzed the TCGA gene expression data to determine whether elements of the single-cell self-renewal gene expression signature that we identified are associated with survival in AML.