RNA-seq analysis of transcriptomic changes in control (shLuc) and GEM knockdown (shGem) bone marrow LSK cells

Cancers develop from the accumulation of somatic mutations, yet it remains unclear how oncogenic lesions cooperate to drive cancer progression. Using a mouse model harboring NRasG12D and EZH2 mutations that recapitulates leukemic progression, we employed single-cell RNA sequencing to map cellular composition and gene expression alterations in healthy or diseased bone marrows during leukemogenesis. At cellular level, NRasG12D induces myeloid-biased differentiation and EZH2-deficiency impairs myeloid cell maturation, whereas they cooperate to promote myeloid expansion with dysregulated transcriptional programs. At gene level, NRasG12D and EZH2-deficiency independently and synergistically control gene expression in single cells. We integrated results from histopathology, leukemia repopulation, and leukemia-initiating cell assays to validate transcriptome-based cellular profiles. We used this resource to relate developmental hierarchies to leukemia phenotypes, evaluated oncogenic cooperation at single-cell and single-gene levels, and identified GEM as a new regulator of leukemia-initiating cells. Our studies establish an integrative approach to deconvolute cancer evolution in vivo. Overall design: Lineage negative cells were transduced with shRNA virus against luciferase (shLuc) or GEM (shGem). Positively transduced cells (GFP+) were sorted by FACS 72 hours post-transduction and subjected to RNA-seq library preparation. RNA-seq library was prepared using the SMARTer stranded pico input total RNA-seq kit (Takara)