Anti-apoptotic A1 is not essential for lymphoma development in Eµ-Myc mice but helps sustain transplanted Eµ-Myc tumour cells

Abstract

The transcription factor c-MYC regulates a multiplicity of genes involved in cellular growth, proliferation, metabolism and DNA damage response and its overexpression is a hallmark of many tumours. Since MYC promotes apoptosis under conditions of stress, such as limited availability of nutrients or cytokines, MYC-driven cells are very much dependent on signals that inhibit cell death. Stress signals trigger apoptosis via the pathway regulated by opposing fractions of the BCL-2 protein family and previous genetic studies have shown that the development of B lymphoid tumours in Eµ-Myc mice is critically dependent on expression of pro-survival BCL-2 relatives MCL-1, BCL-W and, to a lesser extent, BCL-X_L, but not BCL-2 itself, and that sustained growth of these lymphomas is dependent on MCL-1. Using recently developed mice that lack expression of all three functional pro-survival A1 genes, we show here that the kinetics of lymphoma development in Eµ-Myc mice and the competitive repopulation capacity of Eµ-Myc haemopoietic stem and progenitor cells is unaffected by the absence of A1. However, conditional loss of a single remaining functional A1 gene from transplanted A1-a^-/-A1-b ^fl/fl A1-c^-/- Eµ-Myc lymphomas slowed their expansion, significantly extending the life of the transplant recipients. Thus, A1 contributes to the survival of malignant Eµ-Myc-driven B lymphoid cells. These results strengthen the case for BFL-1, the human homologue of A1, being a valid target for drug development for MYC-driven tumours.

Fig. 1

Loss of A1 does not perturb lymphomagenesis in Eµ-Myc mice. a Kaplan–Meier survival curves for A1^+/+ Eµ-Myc (n = 27, median survival of 92 days), A1^+/− Eµ-Myc (n = 64, median survival of 95 days) and A1^−/− Eµ-Myc (n = 49, median survival of 94 days) mice. Data for A1-1 and A1-2 cohorts (Supplementary Figure S2a) have been pooled. The difference in survival between the genotypes was not significant (log⁻rank test). b Stacked bar graph showing the percentages of pro/pre-B (B220⁺sIg^-), B (B220⁺sIg⁺) and mixed pre-B/B cell lymphomas observed for A1^+/+ Eµ-Myc (n = 21), A1^+/− Eµ-Myc (n = 31) and A1^-/- Eµ-Myc (n = 36) mice. c Kaplan–Meier survival curves for pro/pre-B and B lymphoma development in A1^+/+ Eµ-Myc (n = 24), A1^+/− Eµ-Myc (n = 34) and A1^−/− Eµ-Myc (n = 40) mice. Only mice for which immunophenotyping was performed, or that survived until the endpoint, are included in these plots. No significant differences were apparent between genotypes in either the incidence (b) or kinetics (c) of pro/preB and B lymphomas. d Scatter plots of white blood cell counts (WBC) and weights of spleen, lymph nodes (inguinal, axillary and brachial lymph nodes) and thymus of autopsied sick A1^+/+ Eµ-Myc, A1^+/− Eµ-Myc and A1^−/− Eµ-Myc mice that had reached ethical endpoint. Bars represent mean ± SEM. No significant differences were found (Student’s T-test)

Fig. 2

Loss of A1 does not perturb the premalignant phenotype in Eµ-Myc mice. Analysis of spleens of healthy A1^+/+, A1^+/−, A1^−/−, A1^+/+ Eµ-Myc, A1^+/− Eµ-Myc and A1^−/− Eµ-Myc mice (n = 8–9 per genotype, sex matched) euthanased at 28–30 days of age. The total cellularity and compositional analysis of B, T and myeloid cells are shown. Data for bone marrow, blood, lymph nodes and thymus are presented in Supplementary Table S1. Bars represent mean ± SEM. * P < 0.05. No significant differences in the pre-neoplastic phenotype were found between A1^+/+ Eµ-Myc and A1^−/− Eµ-Myc mice (Ordinary one-way ANOVA, Tukey’s multiple comparisons test)

Fig. 3

Loss of A1 does not enhance apoptosis of pre-leukaemic pro/pre-B cells in vitro. a Viability (%) of bone marrow B220⁺sIg^- cells obtained by FACS sorting from healthy (28–30 day-old) A1^+/+ Eµ-Myc and A1^−/− Eµ-Myc mice (n = 3–6 per genotype) and cultured for 48 h without added cytokines. Viability was measured at 0, 4, 8, 24 and 48 h by flow cytometry after staining with propidium iodide (PI) and Annexin V. Bars represent mean viability (PI^- Annexin V⁻) ± SEM. No significant differences were found between A1^+/+ Eµ-Myc (light grey bars) and A1^−/− Eµ-Myc (dark grey bars) cells (multiple Student’s T-tests). b Analysis of A1 expression in bone marrow pro/pre-B (B220⁺sIg⁻) and splenic B (B220⁺sIg⁺) cells obtained by FACS sorting from healthy young A1^+/+, A1^−/−, A1^+/+ Eµ-Myc, and A1^−/− Eµ-Myc mice. Cell lysates (15 µg) from independent mice were run in each lane. Data are representative of two independent experiments. Molecular weight markers are indicated (kD). The immature B cell WEHI-231 line was used as a positive control for A1; treatment with cycloheximide (CHX) resulted in loss of A1 due to its short half-life

Fig. 4

Expression of BCL-2 protein family members in A1^+/+ Eµ-Myc and A1^−/− Eµ-Myc lymphomas and premalignant cells. Western blots were performed on a pro/pre-B cell lymphomas from lymph nodes of sick A1^+/+ Eµ-Myc (lanes 1–4) and A1^−/− Eµ-Myc mice (lanes 5–8) and pre-malignant pro/pre-B cells (MACS-sorted CD19⁺ bone marrow (BM) samples) from healthy A1^+/+ Eµ-Myc and A1^−/− Eµ-Myc mice euthanased at 28–30 days of age (lanes 9–12); b B cell lymphomas from lymph nodes of A1^+/+ Eµ-Myc (lanes 1–4) and A1^−/− Eµ-Myc mice (lanes 5–8). The same premalignant samples and control lysates as in (a) were run to enable comparison between (a) and (b). Lane labels indicate the individual mouse number. WEHI-231 cells served as a positive control for A1 protein, tumour 1/8094 as a positive control for p53 and p19ARF proteins and β-ACTIN as a loading control. Independent blots are separated by a horizontal black line and molecular weight markers (kD) are indicated. Additional lymphomas were analysed (data not shown), in total: 8 pro/pre-B and 8 B A1^+/+Eµ-Myc; 7 pro/pre-B and 6 B A1^−/−Eµ-Myc

Fig. 5

A1^−/− Eµ-Myc haemopoietic stem and progenitor cells are as competitive as A1^+/+ Eµ-Myc cells in reconstituting haemopoiesis. Bone marrow competitive reconstitution experiments were set up using UBC-GFP/Eµ-Myc cells as the competitor. For analysis these can be distinguished by GFP expression from the A1^+/+ Eµ-Myc and A1^−/− Eµ-Myc test bone marrow cells, and by Ly5.2 expression from the Ly5.1 recipients. For each experiment UBC-GFP/Eµ-Myc cells were mixed in a 1:1 ratio with either A1^+/+ Eµ-Myc or A1^−/− Eµ-Myc cells and a total of 3 × 10⁶ cells transplanted into three lethally irradiated Ly5.1 recipients. Six weeks post reconstitution, mice were bled and analysed for GFP, Ly5.2, B220, IgM, IgD, CD4, CD8, Mac1 and Gr1 expression by flow cytometry. The data shown here represents mean ± SEM of 6 independent experiments. Data plotted is % GFP⁻of total Ly5.2⁺, so is a comparison of the test bone marrow vs only UBC-GFP/Eµ-Myc (Ly5.1 cells have been excluded)

Fig. 6

Conditional deletion of floxed A1-b gene in transplanted Eµ-Myc lymphomas enhances survival of transplanted mice. a Survival curves of mice transplanted with Eµ-Myc-driven lymphomas and then treated with tamoxifen or vehicle. Each individual (Ly5.2⁺) tumour from female primary mice (Supplementary Figure S3b) was transplanted intravenously into 6 female Ly5.1⁺ recipients (3 × 10⁶ cells/recipient), three of which received tamoxifen (200 mg/kg body weight) on d5 and d6 post-transplantation and three received vehicle alone. Mice transplanted with A1^+/+ Eµ-Myc (n = 7 independent primary tumours) or A1^+/+ Eµ-MycCreERT2 tumours (n = 7) showed no significant difference in survival between tamoxifen and vehicle treatment. However, mice transplanted with A1^fl/+ Eµ-MycCreERT2 tumours (n = 8) survived significantly longer following tamoxifen treatment (median survival 14 d for vehicle-treated mice vs. 21 d for tamoxifen-treated mice; P = 0.0019, log-rank test), as did mice transplanted with A1^fl/fl Eµ-MycCreERT2 tumours (n = 12) (median survival of 17 d for vehicle-treated mice vs. 23 d for tamoxifen-treated mice; P = 0.0075, log-rank test). Data for A1-1 and A1-2 cohorts have been pooled. (see Supplementary Figure S3C for the data divided into the individual A1 lines). b Analysis of lymphomas arising in the lymph nodes of transplant recipients after treatment with tamoxifen or vehicle. DNA was purified from sorted Ly5.2⁺ lymphoma cells (to exclude any recipient cells, which would not have floxed A1 alleles), after which PCR was performed to confirm the presence of Eµ-Myc, RosaCreERT2, and to determine which A1-b alleles were present (A1-d was also analysed as a control). The number of the primary tumour that was transplanted is indicated above the lanes, and whether the recipient had been treated with tamoxifen or vehicle is indicated as + or − respectively. The survival (in days) of recipients post transplantation is indicated below the blot. In all, 30 tumours were analysed by PCR (11 A1^fl/+ Eµ-Myc CreERT2 and 19 A1^fl/fl Eµ-Myc CreERT2 lymphomas) and in every case deletion of the floxed A1b alleles was very efficient following tamoxifen treatment