Figure 3.

Loss of lymphocyte cell-specific kinase (LCK) negatively effects propagation potential of the T-cell acute lymphoblastic leukemia (T-ALL) cell line MOLT4 and patient-derived xenograft (PDX) L963 in vivo. (A) Volcano plot derived from the functional in vivo screen representing the magnitude of the fold change (log₂) in shRNA abundance derived from leukemia cells isolated from the spleen of PDX L963 on the x-axis. Each dot represents an individual shRNA construct. The y-axis represents the significance in enrichment or depletion of shRNA constructs (log₁₀ scale). Three dots (shLCK#3, shRPL9#1 and shCD19#2) above the blue line are significantly depleted (P<0.05). Bar plot of the normalized shLCK#3 sequencing reads (log₂) in leukemic cells derived from the bone marrow (orange) or spleen (blue) of six individual mice (M1-6), relative to the frequency of these reads before transplantation (green, base line B1-3). (B) Schematic representation of the in vivo competitive outgrowth assay. MOLT4 cells were lentivirally transduced with shNTC (red fluorescent protein, RFP) or shLCK#3 (GFP) and intrafemorally injected into five NSG mice in a 1:1 ratio. Mice were culled once symptomatic and the ratio of RFP : GFP positive human leukemic cells in spleen (n=5), bone marrow (n=3) or liver (n=3) determined by flow cytometry. In all mice, the MOLT4 cells carrying shLCK#3 were outcompeted by shNTC cells during engraftment in spleen, marrow and liver. Student's t-test: *P<0.05, ***P<0.005, ****P<0.001.