Endogenous Nras G12D/+ and G12D/G12D Distinctly Regulate Self-Renewal and Differentiation of Haematopoietic Stem Cells

Abstract 4086

How oncogenes regulate adult stem cells to promote tumorigenesis is poorly understood. We and others previously reported that recipient mice transplanted with Nras G12D/+ or Nras G12D/G12D bone marrow cells develop distinct haemtopoietic malignancies. Mice with G12D/+ cells predominantly develop a myeloproliferative neoplasm (MPN) closely resembling chronic myelomonocytic leukemia (CMML), while animals with G12D/G12D cells develop acute T- or B-cell lymphoblastic leukemia (TALL or BALL) and/or MPN, with varying penetrance, which appear to be regulated by the activity of G12D/G12D haematopoietic stem cells (HSCs). Consistent with this notion, we found that G12D/+ HSCs are required to initiate and maintain CMML-like phenotypes in recipient mice and serve as MPN initiating cells. Therefore, we further investigated how endogenous oncogenic Nras signaling regulates the self-renewal and differentiation of HSCs to promote leukemia development in different lineages of cells.

Here we show that G12D/+ signaling results in moderate hyperproliferation and increased self-renewal of HSCs, promoting expansion of myeloid progenitors and consequently myeloid malignancies. In contrast, G12D/G12D leads to excessive hyperproliferation, decreased self-renewal, and depletion of HSCs, which promote expansion of myeloid and lymphoid progenitors and subsequently malignancies in both compartments. Because leukemia development in Nras G12D/+ and G12D/G12D models is tightly associated with ERK1/2 hyperactivation in haematopoietic stem/progenitor cells (HSPCs), we studied the MEK/ERK signaling in HSCs and their downstream multipotent progenitors (MPPs) using a “HSC phosphor-flow” method we developed. Our data demonstrate that ERK1/2 is hyperactiavated in G12D/+ and G12D/G12D HSCs in a dose-dependent manner, while AKT is not affected in G12D/+ and G12D/G12D HSCs. In contrast, both ERK1/2 and AKT are not changed in G12D/+ and G12D/G12D MPPs. As expected, inhibition of MEK/ERK signaling by AZD6244 (a MEK1 inhibitor) rescues the HSC phenotypes and attenuates myeloproliferative neoplasm phenotypes in G12D/+ and G12D/G12D mice. Mechanistic analysis identifies that a cohort of MAPK pathaway genes regulating cell cycle and signaling are significantly differentially expressed in G12D/+ HSCs compared to control or G12D/G12D HSCs. Unlike the prevailing theory based on Ras overexpression studies, depletion of G12D/G12D HSCs is not associated with overexpression of cell senescence genes. Rather, the Wnt and Notch pathways are significantly downregulated in G12D/G12D but not G12D/+ HSCs. Therefore, we propose that endogenous Nras G12D signaling differentially regulates HSCs self-renewal and differentiation through a dose-dependent hyperactivation of ERK1/2.