Oncogenic Kras Initiates Leukemia in Hematopoietic Stem Cells
Figure 7
Transplanted KrasG12D Flk2− LSK Cells Initiate T-ALLs That Contain Notch1 Mutations
(A) Flow cytometry of thymocytes harvested from moribund recipients of KrasG12D Flk2− LSK cells shows an abnormal accumulation of CD4/CD8 double positive and immature CD8 single positive cells. Spleen histology demonstrates infiltration by monomorphic cells with open chromatin (hematoxylin/eosin).
(B) Primary recipients (n = 5) received 500 KrasG12D Flk2− LSK cells with or without an equal number of WT Flk2− LSK cells after lethal irradiation (950 rad). These primary recipients were euthanized 2–3 mo later, and 106 bone marrow cells or thymocytes were transferred into sublethally irradiated (450 rad) secondary recipients (two per primary mouse). Sublethal irradiation selectively permits transfer of acute leukemia but not KrasG12D HSCs or MPD [13]. Thymocytes, but not bone marrow cells, transferred T-ALL.
(C) Cell lysates from thymocytes and bone marrow of animals euthanized 3 mo after transplantation with KrasG12D or WT HSCs were blotted with an antibody specific for cleaved Notch1. Three independent primary recipients are shown. Sequence analysis demonstrates frameshift mutations near the PEST domain of Notch1 in thymocytes from five of six mice that received KrasG12D HSC, but not in recipients receiving WT HSCs alone (reference sequence from GenBank [http://www.ncbi.nlm.nih.gov/Genbank] accession number NM_008714).