Abstract B34: Single cell RNA sequencing identifies the NRASG12V-mediated AML self-renewal signature.
Authors: Sachs Zohar , LaRue Rebecca S. , Noble Klara , Rathe Susan K. , Sarver Aaron L. , Ha Ngoc A. , Largaespada David A. 
Published: August 2015

Abstract
Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. Using a murine model of AML that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRASG12V), we have found that NRASG12V enforced leukemia self-renewal gene expression signatures and was required to maintain an MLL-AF9 and Myb-dependent leukemia self-renewal gene expression program. In functional assays, NRASG12V was required for leukemia self-renewal independently of its effects on growth and survival (Sachs et al. in revisions). Similarly, recent work has shown that NRASG12V has bimodal, mutually exclusive effects in hematopoietic stem cells (HSCs) providing a proliferation advantage in some HSCs and a self-renewal advantage in others (Li et al. Nature 2013). These data show that mutant NRAS enforces different behaviors in similar cells and suggest that these discrepant effects may be mediated by different NRAS-activated pathways and gene expression pathways. In order to define the mechanisms by which NRASG12V directs self-renewal in AML, we investigated the NRASG12V-mediated transcriptional profile of self-renewing leukemic subgroups and single cells from our AML model. We sorted our primary murine AML into immunophenotypic subgroups and performed bulk cell RNA sequencing on these groups. The Mac-1High group displayed poor self-renewal capacity in colony forming assays and in vivo leukemia transplant assays. In contrast, the Mac-1Low/c-Kit+/Sca-1+ (MLowKS) subgroup was enriched for self-renewal capacity and displayed significantly increased colony forming ability and enhanced in vivo leukemia transplantability. Using principal component analysis and unsupervised hierarchical clustering, we found that each of our immunophenotypic subpopulations were transcriptionally distinct. Using this dataset, we identified the NRASG12V-directed leukemia self-renewal gene expression signature. Gene Set Enrichment Analysis (GSEA, et al. 2005) of this dataset showed that the MLowKS group preferentially expressed previously published transcriptional signatures of leukemia self-renewal. These data also show that NRASG12V enforces distinct gene expression signatures among leukemic subpopulations. Since others have shown that NRASG12V can enforce bimodal behaviors among cells within the same hematopoietic compartment (HSCs, Li et al. Nature 2013), we speculated that the NRASG12V–mediated transcriptional signature critical for leukemia self-renewal may be variable among individual cells. In order to identify that signature and to assess the cell-to-cell heterogeneity of NRASG12V-mediated transcription, we performed single cell RNA sequencing of unsorted and sorted leukemia cells from our murine AML model. In unsupervised hierarchical clustering algorithms, our single cells clustered into five distinct transcriptional signatures, demonstrating that individual leukemia cells are transcriptionally distinct and identifying the NRASG12V-mediated leukemia self renewal signature. Currently, we employ ultra-high parameter flow cytometry (mass cytometry, CyTOF) to identify the cell type specific signaling pathways associated with these discrepant transcriptional signatures. These data identify the NRASG12V-mediated self-renewal gene expression signature at the single cell level and will allow us to identify the specific signaling pathways that activate this signature.

Citation Format: Zohar Sachs, Rebecca S. LaRue, Klara Noble, Susan K. Rathe, Aaron L. Sarver, Ngoc A. Ha, David A. Largaespada. Single cell RNA sequencing identifies the NRASG12V-mediated AML self-renewal signature. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr B34.