Reduced eIF4E function impairs B-cell leukemia without altering normal B-lymphocyte function

Abstract

The cap-binding protein eukaryotic initiation factor 4E (eIF4E) promotes translation of mRNAs associated with proliferation and survival and is an attractive target for cancer therapeutics. Here, we used Eif4e germline and conditional knockout models to assess the impact of reduced Eif4e gene dosage on B-cell leukemogenesis compared to effects on normal pre-B and mature B-cell function. Using a BCR-ABL-driven pre-B-cell leukemia model, we find that loss of one allele of Eif4e impairs transformation and reduces fitness in competition assays in vitro and in vivo. In contrast, reduced Eif4e gene dosage had no significant effect on development of pre-B and mature B cells or on survival or proliferation of non-transformed B lineage cells. These results demonstrate that inhibition of eIF4E could be a new therapeutic tool for pre-B-cell leukemia while preserving development and function of normal B cells.

Keywords: cancer; cell biology; molecular biology.

Graphical abstract

Figure 1

Genetic deletion of Eif4e reduces eIF4E protein expression in p190 cells (A) Established p190 WT and eIF4E^+/- leukemia cells have similar immunophenotype characterized by B220 and CD43 expression. Representative flow cytometry plots of p190 cells. (B) Protein analyses of eIF4E, 4E-BP-1, 4E-BP-2 and actin in WT and Het p190 cells. Relative protein quantification performed by densitometric analysis using ImageJ64 software. Data are expressed as mean ± SEM. Fold change was calculated using actin as loading control and normalized to WT sample for each independent experiment. Significance was calculated using unpaired t test (∗p < 0.05; ∗∗∗∗p < 0.001, n = 7). (C) p190s were generated from WT or Het mice and we used a bicistronic dual Renilla-Firefly luciferase reporter construct to measure cap-dependent translation (Renilla luciferase) relative to cap-independent, Coxsackie virus IRES mediated translation (firefly luciferase) as an internal control. MLN0128 (100 nM) treated cells were used as a control. Data are represented as mean ± SEM. Fold change was calculated using WT vehicle condition. Significance was calculated using a paired one tailed student's t test. (∗p < 0.05; ∗∗p < 0.01; n = 3 or 4 per group). (D) Western blots of eIF4E, 4E-BP-1, 4E-BP-2, and total ERK (tERK) in eIF4E+/+, eIF4E fl/+ and eIF4E fl/fl p190 after 72hr of 4OHT (1 μM) treatment. (E) Relative protein quantification performed by densitometric analysis using ImageJ64 software. Fold change calculated using tERK as loading control. Data are expressed as mean ± SEM. Significance was calculated using paired t test (∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.001, n = 4–6 per group).

Figure 2

eIF4E^+/- and eIF4E^fl/+ p190 have a growth disadvantage (A) Experimental scheme of competitive outgrowth assay of WT or eIF4E^+/- p190 BCR-ABL. (B) Growth of p190 cells was monitored in vitro over 9 days. Percentage of hCD4 and GFP marker positive cells were measured with flow cytometry. Data are expressed as mean ± SEM. (∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 two-way ANOVA, n = 3 per genotype). (C) Log of ratio of hCD4+/GFP+ was measured by flow cytometry. Data are expressed as mean ± SEM. (∗p < 0.05, ∗∗p < 0.01, one way ANOVA, n = 9 or 10 mice/group). Representative flow cytometry plots of bone marrow analyzed for frequency of WT and Het leukemia cells before and after injection of an equal ratio of hCD4 WT and GFP Het p190. (D) Experimental scheme of maintenance assay of WT or eIF4E fl/+ p190 BCR-ABL with 100% marker positivity. (E) Competitive growth assay of established hCD4 and GFP p190 eIF4E+/+ Cre+ and eIF4E fl/+ Cre+ treated with 1 μM 4OHT. Percentage of hCD4 and GFP marker positive cells were measured by flow cytometry. Data are expressed as mean ± SEM. (∗∗∗∗p < 0.0001 two-way ANOVA, n = 6–10/each combination).

Figure 3

Functional analyses of eIF4E+/- mouse splenic B cells (A) Protein analyses of eIF4E, 4E-BP-2, 4E-BP-1 and actin in purified WT or eIF4E^+/- B cells after 16 hr of LPS + IL-4 stimulation. Relative protein quantification performed by densitometric analysis using ImageJ64 software. Data are expressed as mean ± SEM. (∗p < 0.05, ∗∗∗p < 0.001, unpaired one-tailed student's t-test, n = 3 or 4 per group). (B) Renilla-Firefly luciferase cap-dependent translation of WT and eIF4E^+/- B cells stimulated with LPS + IL-4 for 48hr. Data are expressed as mean ratio of Renilla/Firefly +/− SEM. Significance was calculated using a paired one tailed student's t test. (∗∗p < 0.01). (C) Flow cytometry histogram of CFSE staining of WT and eIF4E^+/- B cells in presence of anti-IgM and IL-4. Similar results were obtained in a repeat experiment. (D) Percentage of CFSE-low WT and eIF4E^+/- B cells stimulated with αCD40+IL-4, 5 μg/mL LPS + IL-4, or 0.5 μg/mL LPS + IL-4 and treated with indicated concentrations of rapamycin was measured at 72 hr by flow cytometry. Similar results were obtained in a repeat experiment.

Figure 4

B cell survival and proliferation is not affected by deletion of Eif4e (A) Protein analyses of eIF4E, 4E-BP-1 and tERK in purified splenic B cells from eIF4E^+/+, eIF4E^fl/+ after 96hr of 4OHT treatment in the presence of BAFF survival factor. Fold change calculated using tERK as loading control. Data are expressed as mean ± SEM. (actual p value is shown in graph, paired one-tailed t test, n = 4 per group). (B) Competitive survival assay of purified eIF4E^+/+, eIF4E^fl/+ splenic B cells labeled with or without CFSE in presence of BAFF 100ng/mL. Data are expressed as percentage of positive cells +/− SEM., n = 3–5 per group. (C) Competitive proliferation assay of purified eIF4E^+/+, eIF4E^fl/+ B cells labeled with cell division tracking dyes CFSE or FAR RED (630nM). Following 48hr culture in presence or absence of 4OHT and with BAFF to maintain survival, cells were stimulated to proliferate with 10 μg/mL anti-IgM and 10 ng/mL IL-4. Data are expressed as percentage of dividing cells +/− SEM. (n = 3 for each combination).