Single-cell proteomics characterization of bone marrow hematopoiesis with distinct Ras pathway lesions

Aberrantly elevated Ras signals, triggered by various distinct genetic mutations, are frequent features in myeloid leukemias. Normal hematopoiesis requires perpetual and balanced production of different blood cell lineages by multipotent hematopoietic stem cells (HSCs). Stem and progenitor cells combine dormancy with proliferative drive and require finely tuned metabolism and protein translation rates. Because of the scarcity of stem cells, it has remained largely unknown how aberrantly elevated Ras signals may impact frequency, lineage potential, and quiescent metabolism in rare HSCs. Using single-cell proteomics and computational analyses, we characterized the effects of induced oncogenic mutant KRasG12D or overexpression of the Ras activator RasGRP1, compared with normal native hematopoiesis. The 2 Ras pathway lesions drive shared profound skewing toward and expansion of mature myeloid cells. The resolution of cytometry by time of flight unmasked opposing patterns for the HSC and progenitor compartments: overexpression of RasGRP1 induced expansion of both subsets, whereas KRASG12D resulted in depletion. By combining spectral flow with SCENITH (Single-Cell ENergetIc metabolism by profiling Translation inHibition), a method to quantitate protein translation as a proxy for metabolic state, we first corroborated that immature cells display low metabolic SCENITH rates. Both RasGRP1 and KRASG12D drive elevated, mean SCENITH signals in immature hematopoietic cells. However, RasGRP1-overexpressing stem cells retain a metabolically quiescent cell fraction, whereas this fraction is incompatible with KRASG12D. Our temporal proteomics and metabolomics data sets provide mechanistic insights into altered hematopoiesis at single-cell resolution and support the idea that the exact identity and duration of signals from Ras lesions has profound impacts on stem cell maintenance and lineage potential.