Loss Of Tumor Suppressor BTG1 Enhances ATF4 Function and Promotes Cell Survival

We and others have shown that the B cell Translocation Gene 1 (BTG1) locus is affected by genomic deletions in 9% of pediatric acute lymphoblastic leukemia (ALL) patients. The fact that multiple subclones carrying distinct deletions can be present in individual patients suggests that interfering with normal BTG1 function provides a selective growth advantage to leukemic cells. However, it remains unclear how loss of BTG1 promotes clonal outgrowth.

We detected an up to 15-fold increases of BTG1 expression when lymphoid cells were exposed to various challenge conditions, including nutrient limitation and ER stress induction. To test for a functional role for BTG1 in the cellular response to stress, we cultured BTG1 knockout cells in medium without glucose or amino acid (Figure 1) and found that BTG1 knockout cells show a 20-30% improved survival rate as compared to wildtype cells.

Figure 1

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BTG1 knockout cells are resistant to Asparaginase treatment.

Figure 1

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BTG1 knockout cells are resistant to Asparaginase treatment.

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As Activating Transcription Factor 4 (ATF4) is a master regulator of cellular stress signaling, we hypothesized that the improved survival after BTG1 loss is regulated via ATF4. By immunoprecipitation experiments, we showed that BTG1 complexes with ATF4. In addition, co-expression of BTG1 attenuates ATF4 transcriptional activity on target gene promoters and suppresses both recombinant and endogenous ATF4 function in these promoter reporter assays (Figure 2).

Figure 2

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BTG1 attenuates ATF4 transcriptional activity.

Figure 2

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BTG1 attenuates ATF4 transcriptional activity.

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Although BTG1 possesses no catalytic activity, it functions as a transcriptional co-regulator that acts by recruiting Protein Arginine Methyl Transferase 1 (PRMT1) to transcription factor complexes. By in vitro methylation assays with purified proteins we showed that ATF4 is directly methylated by PRMT1 on a single arginine residue. In addition we found that the PRMT1 interacting domain in BTG1, while dispensable for the BTG1-ATF4 interaction, is essential for the BTG1 mediated suppression of ATF4 function. In search for additional evidence for the functional interaction between BTG1 and ATF4 we performed global expression analysis on murine cells expressing the B cell marker B220. This revealed a significant deregulation of ATF4 target genes in BTG1 knockout cells when compared to wildtype cells.

Together, our data indicate that BTG1 suppresses activation of ATF4 in response to cellular stress. Loss of BTG1 function, as it occurs during leukemia development, enhances ATF4 activity, thereby promoting cell survival under cellular stress conditions such as nutrient deprivation or ER stress. Leukemic cells carrying BTG1 deletions may thus benefit from this increased resistance to cellular stress, not only during leukemia development but also during treatment. Hence, targeting the ATF4 stress response pathway may prevent relapse of therapy-resistant leukemic clones.

Cells were treated with 2 IU/L Asparaginase for 24 hours. After treatment, cell viability was measured using an MTT assay. The average of 4 independent experiments is plotted with error bars representing the standard error of the mean.

A luciferase reporter gene controlled by the ATF4 responsive ASNS promoter region was transfected into HEK293 cells. Asparaginase treatment induces endogenous ATF4 expression, which results in an increase in luciferase signal (Mock transfected cells). Co-expression of BTG1 represses both endogenous ATF4 activity as well as ectopically expressed ATF4 activity as detected by a decrease in luciferase signal. The average of 2 independent experiments is plotted with error bars representing the standard deviation.