The expansion of T-cells and hematopoietic progenitors as a result of overexpression of the lymphoblastic leukemia gene, Lyl1 can support leukemia formation

Abstract

This study investigates the function of the lymphoblastic leukemia gene, Lyl1 in the hematopoietic system and its oncogenic potential in the development of leukemia. Overexpression of Lyl1 in mouse bone marrow cells caused T-cell increase in the peripheral blood and expansion of the hematopoietic progenitors in culture and in the bone marrow. These observations were the result of increased proliferation and suppressed apoptosis of the progenitor cells caused by the Lyl1-overexpression. Our studies present substantial evidence supporting the secondary, pro-leukemic effect of Lyl1 in early hematopoietic progenitors with the potential to cause expansion of malignant cells with a stem/early progenitor-like phenotype.

Figure 1. Lyl1-overexpression outline

Schematic representation of the MSCV retroviral constructs and the overexpression experimental design. Peripheral blood, pre-cleared from red blood cells, was treated with a cocktail of antibodies and then analyzed by flow cytometry. The donor-derived white blood cells (CD45.2 positive) were separated based on their GFP positivity and then further fractionated as myeloid, B- and T-cells.

Figure 2. Effect of Lyl1-overexpression on the peripheral blood

Peripheral blood lineage analyses of mice transplanted with control (GFP-transduced) or Lyl1–transduced (Lyl1 + GFP) Sca-1⁺ bone marrow cells. The blood was analyzed by flow cytometry every 2 or 4 weeks starting 4 weeks (for total GFP expression) or 12 weeks (for peripheral blood lineage compartments) until 30 weeks post-transplantation. The graphs represent the analysis of the GFP⁺ donor-derived white blood cells: A) GFP⁺ donor-derived white blood cells, represented as a percent of the total white blood cells; B-D) lineage distribution within the GFP⁺ donor-derived cells: T-cells (B), B-cells (C) and Myeloid (D). The error bars represent the average deviation of 3 to 5 mice. The p value for the GFP expression profile was calculated using Graph Pad Prism Version 5 software and a third order polynomial curve fit. * p<0.05; ** p<0.06, determined by unpaired, two tailed t test.

Figure 3. Analyses of cultured bone marrow cells transduced with Lyl1

A) Sca-1⁺ bone marrow cells, transduced with GFP or Lyl1-GFP were cultured as described. After 48 hrs, the KSL population was measured and single Sca-1⁺, GFP⁺ cells were sorted into each well of a 96-well plate containing M3434 MethoCult medium. B) After 14 days of incubation, each colony larger than 2 mm was analyzed for Sca-1 and c-Kit expression using flow cytometry. Cells, double positive for c-Kit and Sca-1 were designated as KS. Total of 26 (GFP) and 33 (Lyl1) colonies, from 2 separate experiments were analyzed. Each data entry represents the percentage of KS cells for each colony. The calculated medians are 42.9 (GFP) and 88.4 (Lyl1). C) Graphical representation of the KSL compartment as a percentage of the GFP⁺ cells from three independent experiments and total of 5 (GFP) and 6 (Lyl1) biological replicates. The error bars are the corresponding calculated SEM. The p values were derived using two tailed, unpaired t test.

Figure 4. Effect of Lyl1-overexpression on the KSL and CLP progenitors in the mouse bone marrow

A) Bone marrow derived from mice transplanted with Sca-1⁺ bone marrow cells transduced with GFP- or Lyl1-expressing MSCV virus was isolated and the KSL and CLP progenitors analyzed as follow: B) the KSL population was derived from the GFP⁺, Lineage^- cells which were further separated as double positive for Sca-1 and c-Kit; the CLP population, also double positive for Sca-1 and c-Kit was derived from the IL7Rα⁺, Lineage^-, GFP⁺ cells. C) The bar graphs represent the KSL or the CLP populations as a percentage of the total GFP⁺ bone marrow cells. The error bars represent the SEM derived from three to five biological replicates.

Figure 5. Role of Lyl1 in cell proliferation, apoptosis, and Sca-1 and c-Kit expression

A) Sca-1⁺ mouse bone marrow cells, transduced with GFP-only or Lyl1 and GFP were transplanted into recipient mice or cultured for 48 or 96 hrs as described. The cultured cells were used in the following two ways: B) GFP⁺ cells were sorted into lysis buffer for RNA isolation which consequently was used for cDNA synthesis. The cDNA was then used as a template in Real-Time qPCR expression analyses of Lyl1, Sca-1, c-Kit and 18S as a control. Cycle thresholds (Ct) specific for each gene were normalized to the 18S Ct by subtraction (ΔCt). Next the ΔCts from the Lyl1 set were subtracted from the ΔCts of the GFP set, thus obtaining the ΔΔCt. The fold change was calculated from the formula 2⌃(ΔΔCt). The bar graph represents the average values obtained from triplicates of three biological replicates. For each gene the GFP value has been set to 1. The error bars are the corresponding SEM. C) The cultured cells were first incubated with anti-Sca-1-PE conjugated antibody and then with AnnexinV-APC conjugate following company protocol. The AnnexinV⁺ cells from three independent experiments were measured and the average plotted as a percent of the GFP⁺, Sca-1⁺ cells. The error bars represent the SEM. The p value was derived using two tailed, unpaired t test. D) The average BrdU⁺, donor KSLs were presented graphically as a percent of the total donor KSLs. The averages were obtained from 4 (GFP) and 6 (Lyl1) biological replicates and two independent experiments. The error bars represent the SEM.

Figure 6. Development of NICD driven leukemia in the absence of Lyl1

A) Wild-type and Lyl1^-/- Sca-1⁺ mouse bone marrow cells, transduced with GFP-only or NICD and GFP were transplanted into recipient mice or cultured for 96 hrs as described. B) The percent of CD4 and CD8 positive cells in the transduced donor derived peripheral blood was analyzed using flow cytometry. The averages from 3 to 7 specimens with the corresponding SEM were graphed. C) The number of the apoptotic cultured progenitors was measured using AnnexinV staining as shown previously. The AnnexinV⁺ cells were presented as a percent of the transduced Sca-1⁺ cells. The p values were derived using two tailed, unpaired t test.