Murine NOTCH1 leukemias lacking both Pten and Ink4a/Arf remain dependent on Notch signaling and are GSI sensitive. (A) PCR analysis confirming homozygous deletion of both Ink4a/Arf (upper panel) and Pten (bottom panel) loci in L1601P-ΔPEST leukemias derived from Pten^f/f Ink4a/Arf^f/f Mx1-Cre bone marrow (n = 7). Analysis of 5-FU–treated bone marrow revealed a subpopulation of progenitors already deleted for Ink4a/Arf and/or Pten prior to retroviral transduction. (B) In vitro proliferation analysis of primary mouse L1601P-ΔPEST leukemias on Pten, Ink4a/Arf double-null background treated with GSI as in Figure 2A. Primary leukemia cells from 5 individual mice were assayed. Only gated GFP⁺ events are depicted. (C) In vivo assay for GSI sensitivity confirms in vitro results. Splenic tumor cells from a single Pten, Ink4a/Arf double-null L1601P-ΔPEST primary leukemia were serially transplanted by tail vein injection into secondary recipients. At day 3 after transplantation, mice were treated by intraperitoneal injection with GSI (100 mg/kg per day DAPT) for 1 day (n = 5) or 2 days (n = 3), or DMSO vehicle only (n = 4). Animals were then killed on day 3 after treatment initiation (day 6 after transplantation) and extent of tumor infiltration in liver was assessed by automated image analysis of H&E histology. Each data point represents a separately imaged histologic field; 3 fields were examined per mouse. Significantly fewer blasts were observed in GSI-treated mice (P < .001; 1-way analysis of variance with posttest linear trend analysis).