Mutant N-ras Induces Myeloproliferative Disorders and Apoptosis in Bone Marrow Repopulated Mice

Abstract

Mutations that activate the N-ras oncogene are among the most frequently detected genetic alterations in human acute myeloid leukemias (AMLs), Philadelphia chromosome-negative myeloproliferative disorders (MPDs), and myelodysplastic syndromes (MDSs). However, because N-ras has not been shown to induce these disorders in an in vivo model, the role of N-ras in the evolution of myeloid leukemia is unclear. To investigate the potential of N-ras to induce myeloid leukemia, lethally irradiated mice were reconstituted with bone marrow (BM) cells infected with a retroviral vector carrying activated N-ras. Approximately 60% of these mice developed hematopoietic disorders, including severe MPDs resembling human chronic myelogenous leukemia (CML) or AML with differentiation (French-American-British [FAB] classification M2). Other reconstituted mice succumbed to hematopoietic defects that were pathologically similar to human MDSs. The latter disorders appeared to be due to a myeloid impairment that was demonstrated by enumeration of day-12 colony-forming units-spleen (CFU-S) and by in vitro colony assays. A high level of apoptosis associated with thymic atrophy and peripheral blood (PB) lymphopenia was also evident in N-rasreconstituted mice. Our results are consistent with a model in which antiproliferative effects are a primary consequence of N-rasmutations and secondary transforming events are necessary for the development of myeloid leukemia. This is the first report of an in vivo model for N-ras induced MPD and leukemia.

Involvement of Gpr125 in the myeloid sarcoma formation induced by cooperatingMLL/AF10(OM-LZ)and oncogenicKRASin a mouse bone marrow transplantation model

“…In our study, we demonstrated that KRAS G12C alone could not immortalize murine BM cells, which was consistent with the previous studies that oncogenic N-/KRAS needed to cooperate with additional mutations to induce hematological malignancies. 22,23 In contrast, BM cells were readily immortalized by MLL/AF10(OM-LZ) alone 18 or combined KRAS G12C and MLL/AF10(OM-LZ). This result showed that KRAS G12C was not necessary for the MLL/AF10(OM-LZ) cell immortalization.…”

Understanding and Targeting Epigenetic Alterations in Acquired Bone Marrow Failure

“…Expression of oncogenic NRasG12D and an empty shRNA vector control led to a progressive myeloproliferative disorder as previously described (MacKenzie et al, 1999). In contrast, expression of NRasG12D in combination with validated mASXL1 knockdown vectors resulted in accelerated myeloproliferation and impaired survival compared with mice transplanted with NRasG12D/EV (median survival 0.8 month for ASXL1 shRNA versus 3 months for control shRNA vector: p < 0.005; Figure 7C) We also noted impaired survival with an independent mASXL1 shRNA construct (p < 0.01; Figure S6B) Mice transplanted with NRasG12D/Asxl1…”

“…Previous studies have shown that hematopoietic cells from mice expressing oncogenic Ras alleles or other mutations that activate kinase signaling pathways do not exhibit increased self-renewal in colony replating assays (Braun et al, 2004;MacKenzie et al, 1999). This is in contrast to the immortalization of hematopoietic cells in vitro seen with expression of MLL-AF9 (Somervaille and Cleary, 2006) or deletion of Tet2 (Moran-Crusio et al, 2011).…”

Mechanistic and Preclinical Insights from Mouse Models of Hematologic Cancer Characterized by Hyperactive Ras

“…1), which was facilitated by development of the murine stem cell virus (MSCV) vector. In one study, 60% of irradiated recipient mice injected with bone marrow cells that had been transduced with a retrovirus expressing an oncogenic Nras allele developed a spectrum of myeloid malignancies (MacKenzie et al 1999). However, the phenotypic variability, incomplete penetrance, and prolonged latency combined with both impaired in vitro proliferation and a high rate of apoptosis in Nras-infected cells provided an early indication that levels of oncogenic Ras expression strongly modulate cellular phenotypes.…”

“…The robust ability of oncogenic Hras to induce myeloid malignancies despite the very low frequency of HRAS mutations in patient samples (Ward et al 2012), coupled with the observed effects of viral titer, suggests that the superphysiological levels of Ras expressed in these systems strongly influence disease phenotypes. Importantly, the hematologic cancers that emerge from retroviral transduction/transplantation experiments invariably exhibit clonal retroviral integrations (MacKenzie et al 1999;Parikh et al 2006Parikh et al , 2007, arguing that a specific level of oncogenic Ras protein expression is strongly selected for, or that misregulation of genes near the integration site or other cooperating somatic mutations are required to induce leukemia in vivo. Several recent studies have deployed modified retroviral transduction/transplantation or transgenic models to examine Ras membrane trafficking, the roles of different effector pathways in leukemogenesis, the ability of oncogenic Ras expression to cooperate with other mutations to generate myeloid malignancies, and to perform preclinical testing of various therapeutic strategies.…”