Single cell RNA-seq reveals developmental plasticity with coexisting oncogenic and immune evasion programs in ETP-ALL.

Lineage plasticity and stemness have been invoked as the cause of therapy resistance in cancer, as these flexible states allow cancer cells to de-differentiate and alter their dependencies. We investigated such resistance mechanisms in relapsed / refractory early T-cell progenitor acute lymphoblastic leukemia carrying activating NOTCH1 mutations, by full-length single cell RNA sequencing of malignant and microenvironmental cells. We identified two highly distinct stem-like states that critically differ in their cell-cycle and oncogenic signaling. Fast-cycling stem-like leukemia cells demonstrate Notch activation and are effectively eliminated in patients by Notch inhibition, while slow cycling stem-like cells are Notch-independent but rather rely on PI3K signaling, likely explaining the poor efficacy of Notch inhibition in this disease. Remarkably, we find that both stem-like states can differentiate into a more mature leukemia state with prominent immune-modulatory functions, including high expression of the LGALS9 checkpoint molecule. These cells promote an immunosuppressive leukemia ecosystem with clonal accumulation of dysfunctional CD8+ T cells that express HAVCR2, the cognate receptor for LGALS9. Our study identifies complex interactions between signaling programs, cellular plasticity and immune programs that characterize T-ALL and illustrates the multi-dimensionality of tumor heterogeneity. In this scenario, combination therapies targeting diverse oncogenic states and the immune ecosystem appear most promising to successfully eliminate tumor cells that escape treatment through co-existing transcriptional programs.